The Glasswing Paradox & Research at the Smithsonian in Panama

How does an animal become invisible? Enter the paradox of the Glasswing butterfly. As the name implies, these butterflies have transparent regions in their wings, engendering a common notion that they are “invisible” in the context of camouflage to avoid predators.

 The “Glasswing” butterfly  Greta oto

The “Glasswing” butterfly Greta oto

However, these butterflies can contain striking orange and iridescent patterns in the wings and numerous other species are known to mimic their wing patterns, highlighting the fact that these butterflies are in fact toxic, as they sequester noxious chemicals called pyrrolizidine alkaloids. Therefore, the bright colors serve as a warning signal to would-be predators such as birds.

The glasswing butterfly Greta oto was also found to harbor randomly sized nanopillars of high aspect ratio, which enables omnidirectional anti-reflection properties. More recently, another transparent-winged butterfly in the genus Chorinea was found to have dome-shaped chitin nanostructures on the wing membrane that result in wavelength-selective anti-reflective and angle-independent transmission. The aforementioned anti-reflective nanostructures found in nature are proving to be quite rewarding for applications in biomimetics and biophotonics, such as solar cells, anti-glare glasses and optical implant devices.

 The role of random nanostructures for the omnidirectional anti-reflection properties of the glasswing butterfly by  Siddique et al. 2015

The role of random nanostructures for the omnidirectional anti-reflection properties of the glasswing butterfly by Siddique et al. 2015

But before diving more into transparency, let’s take a step back for a moment to consider where the color in a butterfly wing comes from in the first place. The primary unit for color in Lepidoptera is the wing scale cell and the underlying mechanism for a particular color can be due to either pigmentation from a biochemical pathway, or due to the physical architecture of scales manipulating wavelengths of light, known as structural color. To better understand processes underlying structural scale modifications, my dissertation has focused on a unique coloration strategy: wing transparency within butterflies and moths (Lepidoptera). Numerous species of Lepidoptera develop wings that allow light to pass through so that objects behind them can be distinctly seen, which has engendered a common notion that these species are “invisible” in the context of camouflage to go undetected by predators. However, my lab and collaborators hypothesize that transparency is a much more complex coloration strategy, playing roles in visual communication through light polarization and iridescence. Terrestrial transparency also entails challenging optical requirements and the morphological, physiological, and genetic mechanisms involved are virtually unknown.

 First visit to the Smithsonian Tropical Research Institute, Gamboa Panama

First visit to the Smithsonian Tropical Research Institute, Gamboa Panama

To investigate the development of transparent species endemic to the Neotropics, I realized that it was critical to obtain living specimens at various life stages. Furthermore, experiments in developmental biology often require access to tissue at precisely known time-points, and therefore being able to raise the organism of interest while preparing for trials is necessary. Therefore, I turned my sights to the Smithsonian Tropical Research Institute (STRI) located in Gamboa, Panama. The facilities at STRI look incredible, if not a touch out of place. Nestled into a small sleepy town in the rainforest, STRI has recently been upgraded to a building containing state-of-the-art molecular laboratories. This field site has also been utilized for many years by researchers investigating color pattern formation in Heliconius butterflies, who have established stellar insectaries and have knowledgeable staff on site for plant and animal husbandry.

 A pupa of the Glasswing raised at STRI

A pupa of the Glasswing raised at STRI

My goal at STRI was to raise Glassing butterflies, then investigate and experimentally manipulate pupal wings at various developmental stages in order to identify cellular and cytoskeletal scale modifications. During my expedition I was successfully able to collect and established a colony of glasswing butterflies at the local insectary. Taking advantage of the laboratory at STRI, I was able to perform dissections of pupal wings and stain wing tissue with fluorescent markers such as DAPI and phalloidin to visualize nuclei and scale cytoskeletal modifications. Additional tissue was preserved for downstream genomic and RNA experiments. Results thus far indicate that glasswing butterflies become transparent by modifying the ploidy levels in scale cell nuclei, as transparent regions of the wing have smaller and more spaced-out cells, and phalloidin stainings indicate that modifications to F-actin during scale growth may play a role in the peculiar "forked" bristles present on glasswings, which allows light to pass through to the membrane of the wing which harbors anti-reflective nanostructures. This has been a critical first step to employ experiments investigating the development of transparency, including gathering material for comparative transcriptomics and functional modifications via CRISPR-cas9 genome editing. The results from this expedition and future work on the established colony can now feed into comparative analyses, help elucidate the genetic drivers of scale structural complexity, and provide insight into the evolution of terrestrial transparency.

 Iridescent wing patterns at certain angles of light on the wings of transparent species

Iridescent wing patterns at certain angles of light on the wings of transparent species

So, what’s the deal Glasswings? Are you transparent to go unseen? Are you bright to show off warning colors? Perhaps a bit of both? I think it’d be interesting if the dual nature serves to avoid a certain kind of predator under reflected light. Another possibility is that they’re showing off ultraviolet colors as warnings, which would be invisible to us, but clear as day to other animals such as birds, many of which contain opsins in their eyes capable of detecting UV. Either way, they’re a beautiful group of butterflies and it’s a beautiful scientific mystery to (attempt to) solve the evolution and development of transparency. These experiments at STRI would not have been possible without the help of the Tinker Summer Field Research Grant, to which I am very thankful for the opportunity. While this was my first visit the Smithsonian Tropical Research Institute, I have little doubt that it will not be my last.

Jungle Genomics Field Course Announcement

I'm pleased to announce the first ever 'Genomics in the Jungle' field course, which will take place July 22 - August 4! https://fieldprojects.org/course/genomics-in-the-jungle/ 

 My portable lab setup that I recently deployed in Peru to sequence long stretches of ribosomal DNA from plants and arthropods.

My portable lab setup that I recently deployed in Peru to sequence long stretches of ribosomal DNA from plants and arthropods.

In the past I have worked with Field Projects International to co-run a two week Field Entomology course, and this time around we will aim to apply portable technology to a field course in the Peruvian Amazon.

Here is a short description for the upcoming trip below:

Course Description

Biological research is turning to genetic research methods for a deeper look into the factors that encode behavior and physiology. We use genetic techniques to determine species delimitations, define populations, understand mating systems, explain behavioral differences in foraging efficiency, screen for disease, conduct paternity studies, evaluate immune status and functioning, and explore microbiome diversity… and these are just a few examples of the full breadth of the field as applied to wildlife biology.  The field of genetics is revolutionizing biological research, and in the past few years we have even witnessed the successful deployment of instruments that enable molecular work to be conducted ‘on-the-fly’ and in the field. These new tools are minimizing the hassles and barriers associated with transporting samples around the world to distant labs that possess the equipment and resources to extract, amplify, and sequence DNA. In many ways, this new technology is democratizing wildlife research by empowering field scientists all around the world with genetic tools to directly advance their research and conservation initiatives.

This course will take you to the Peruvian Amazon, where you will learn how field research is conducted, assist in sample collection, and then actually extract, amplify, sequence, and interpret genetic data to answer several practical research questions about wildlife ecology and natural history.  It will take place at the Inkaterra Field Guides Station, which is the site of the Green Lab, the world’s first tropical rainforest molecular genetics laboratory. You will go from sample collection to sequence analysis directly in the rainforest. This course is will provide an introduction to next-generation primatologists and biologists, who will gain not only the skills requisite for field research but the technical know-how to employ genetic research tools in the field.   

Research Topics

In this course, we will focus on three specific cases in which cutting-edge genomics can help us solve mysteries common to wildlife research in the field. The ultimate goal of all of these projects will be to use a MinION, a USB-sized powerful sequencer that is revolutionizing how we do genomics in some of the craziest places on the planet.

The MinION has been used to:

We will study three specific cases (class size and time permitting):

  1. DNA fingerprinting
  2. The effects of captivity on microbiome diversity
  3. Environmental DNA

We will try to answer a few additional questions with all case studies:

  • Can we use the What’s In My Pot (WIMP) workflow to accurately classify these species to a reference database in real-time for all of the case studies?
  • How many different kinds of samples can we multiplex at one go on a MinION?
  • Does the choice of DNA extraction kit affect the outcome?
  • Does the length of time we let the MinION run for affect the accuracy of our species identifications?

If this sounds interesting to you or someone you know, please check out the link for more information https://fieldprojects.org/course/genomics-in-the-jungle/

It's an exciting time to be a biologist!

From Snakes to Sequences in 24 Hours: Success!

 

As I type up my notes from the field, I can only imagine what Darwin would think about the advancements in technology used to answer questions about evolutionary biology today...

 

7/11/17 6:00 AM

We hit the ground running after sharing the last post at the airport.  Landing at around 6:00 AM in Quito, Ecuador, we were greeted by our collaborators Lucas Bustamante (Tropical Herping) and Dr. David Salazar (Universidad Indoamerica), threw our gear in the truck, and swung by the University lab to pick up our final crew member, Nicolás Peñafiel. All the equipment and reagents for sequencing appeared to have traveled well on ice packs (at least we hoped so!), so we transferred our frozen material into a cooler and were off to the Chocó rainforest of Ecuador.

 The "boat" responsible for transferring our vehicle across the Canandé river

The "boat" responsible for transferring our vehicle across the Canandé river

7/11/17 3:00 PM

After several hours of driving through bumpy jungle roads and crossing the Canandé river on a questionable boat, we arrived at the small lodge nestled in the Chocó rainforest and rendezvoused with the rest of the team, who arrived a couple days ahead of us, including Alejandro Arteaga, Frank Pichardo, and Cesar Barrio Amorós.

Our goal was to survey the region for unique reptiles and amphibians and use the portable laboratory to sequence DNA right there in the field, so no time to waste! Nico and I unpacked the lab equipment and at around 8:24 PM began a few DNA extractions on a whip snake sample using a salt extraction protocol and the DNeasy kit protocol. Then as night fell, we grabbed our headlamps and were out in search of new targets. After several hours spotting numerous frogs and geckos, Frank stumbled upon a gorgeous eyelash viper just off trail.

 Eyelash viper posing on the MinION DNA sequencer

Eyelash viper posing on the MinION DNA sequencer

 A beautiful shot of our viper next to the MinION by Lucas Bustamante

A beautiful shot of our viper next to the MinION by Lucas Bustamante

7/11/17 11:34 PM

Back at the lodge that evening, we prepped a PCR run using the miniPCR and let it go overnight. Time for some rest.

7/12/17 10:15 AM

While Alejandro took digital white background images of the animals from last night, Nico and I made a gel to visualize the PCR product. The small electrophoresis chamber Nico brought from the lab worked well, but unfortunately my small UV flashlight was unable to really pick up fluorescence in the gel to visualize amplicons. I had tested this out briefly in the US before the trip and was able to pick up bands in a pitch black room, but I think the UV light wasn't quite strong enough under field conditions. Some of the bands appeared to be there which was encouraging, but next time I think it would be beneficial to tinker with a small transilluminator like the one also made by miniPCR.

7/12/17 11:30 AM

Alejandro wrapped up processing his specimens and extracting a small amount of blood or tail tissue from each, so I got to work extracting DNA for the eyelash viper and dwarf geckos. The extraction process with the DNeasy kit is easy (as the name implies!); as for equipment just requires a small centrifuge and the reagents can be stored at room temperature. After about an hour we had our fresh DNA samples.

 Part of the DNA extraction and PCR set-up at our field site.

Part of the DNA extraction and PCR set-up at our field site.

1:07 PM

Next I got started with a new round of PCR using primers for genes for 16S, cytb and ND4. These primers don't all necessarily have the same PCR conditions (such as annealing temperature) but for the sake of time and limitations with one miniPCR, I ran them together under the same settings.

3:25 PM

After a couple hours of PCR cycles, it was time for the second PCR barcding step. I used barcodes 1 through 8 for the samples and ran the new PCR protocol.

4:30 PM

Finally, it was time to start the library preparation for the nanopore sequencer using the SQK-LSK 1D kit. This involves the end-prep, adapter ligation and bead cleanup. Next it was time to prime the flow cell and load the sample.

 The MinION (top) and miniPCR (bottom) make a great portable duo!

The MinION (top) and miniPCR (bottom) make a great portable duo!

6:24 PM

I clicked "execute" for the MinKNOW software, began the sequencing run and said 'hold on to your butts' (one of my favorite quotes from Jurassic Park).

 The MinION sequencer glowing red and blue as it runs off the power of my laptop.

The MinION sequencer glowing red and blue as it runs off the power of my laptop.

 Some of the sequence data produced from the MinION sequencer.

Some of the sequence data produced from the MinION sequencer.

7:20 PM

After about an hour, I stopped the run after 16,484 reads had been generated, and ran the data through the Albacore program to demultiplex the reads into their individual barcode folders. I then took a peak in the barcodes and was excited to see the read lengths looked correct, so I downloaded barcode 1 and passed it along to Alejandro's laptop. Barcode 1 was the 16S sequence for the eyelash viper and Alejandro had a nice reference database on his laptop to compare the nanopore sequence to. After a few minutes of tinkering, Ale said "yes, it falls out with Bothriechis schlegelii!". This was it! The nanopore barcode was a complete match to the correct species!

 BLAST hit result using a consensus read from the nanopore 16S barcode, which is a 98% match to the correct viper species. It will be interesting to see if the 2% difference is due to individual genetic variation, or if the difference is due to nanopore sequence error, which will be verified with Sanger sequencing of the same individual.

BLAST hit result using a consensus read from the nanopore 16S barcode, which is a 98% match to the correct viper species. It will be interesting to see if the 2% difference is due to individual genetic variation, or if the difference is due to nanopore sequence error, which will be verified with Sanger sequencing of the same individual.

 Components of the portable lab used on the trip. Left to right: miniPCR sitting atop the Poweradd battery, Vaio laptop with Geneious pro software to visualize sequence data, and the ONT MinION sequencer powered by the laptop.

Components of the portable lab used on the trip. Left to right: miniPCR sitting atop the Poweradd battery, Vaio laptop with Geneious pro software to visualize sequence data, and the ONT MinION sequencer powered by the laptop.

In less than 24 hours from arriving at the field site and sampling snakes and geckos during our first night in the rainforest, we could verify species identification by creating a nanopore consensus and mapping to a pre-downloaded reference database. Now we are also verifying if some species collected are undescribed back at a lab in Quito, and will have everything Sanger sequenced to verify nanopore quality.

9:30 PM

After getting back from the field, Stefan set out to process the reads. While Geneious is good enough for a quick peek, it cannot deal with Nanopore-specific errors. After some quick tests using reference-based mapping (using bwa mem, samtools, angsd and nanopolish) it was clear we got good consensus sequences for all the 16S and the ND4 genes! However, CytB and COI did not amplify, which we verified on a gel back at Quito university a few days later, likely because we didn't have time to run multiple PCRs under optimal contitions for all the genes. Stefan then worked on tweaking de-novo assembly tools such as Canu and Allele Wrangler, to create consensus sequences without reference bias. All sequences from the field were later processed with Canu, which turned out to work pretty well for amplicon assembly.

 Stefan working on his nanopore bioinformatics in the jungle.

Stefan working on his nanopore bioinformatics in the jungle.

Overall, we believe this is important becuase the Ecuadorian Chocó is a biodiversity hotspot which has lost more than 98% of habitat due in large part  to logging and palm oil agriculture. Rapid sequencing can be a useful  tool to better understand the diversity of life on our planet and use  that information for conservation. Furthermore, researchers in Quito do not have access to a Sanger sequencer let alone Next Generation Sequencing platforms within the country. Thus, Oxford Nanopore MinION sequencing enables them to rapidly process samples without the need to send them off internationally.

 Some of the expedition team members! Left to right: Alejandro, Aaron, Frank and Lucas

Some of the expedition team members! Left to right: Alejandro, Aaron, Frank and Lucas

Right now I'm still feeling a bit of relief and excitement that everything worked in one go in the field. This was an idea that I've been hoping to execute for a while now, and this seemed like the opportune time with a grant funding the project from National Geographic. The work isn't quite done yet, because we are verifying the quality of sequences, performing a few more experiments in lab, and writing all the methods and bioinformatics pipelines up for a publication with our Ecuadorian collaborators. While we quickly and successfully sequenced DNA in the field for correct species identification, this feels like just the beginning!

-Aaron & Stefan