A final round of histology

The last few months I have been glued to the computer working on writing and data analysis, but recently I got a break with some time in the lab. In February, I made a quick trip to Quito to visit our collaborators at Centro Jambatu and pick up some additional samples. Dr. Luis Coloma and his staff at Jambatu are some of the world’s best in breeding endangered frogs like this beautiful Atelopus.

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After picking up the samples and export permits, I headed to the lab in Fort Collins. Following some intense weeks at the microtome and microscope, I now have the image stacks needed to finish up the last 3D models of ear development in our toads.

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New paper in Zoomorphology

Here’s a new paper out titled “Developmental morphology of granular skin glands in pre-metamorphic egg-eating poison frogs”. Its work done in collaboration with Dr. Lauren O’Connell at Harvard, taking the next step from the findings of my PhD dissertation that poison frog mothers provision their offspring with alkaloids.

The study shows that tadpoles develop their glands around the same time that we first saw alkaloids (stage 32-40 of development), so it is likely that the appearance of those glands delimits the point at which the tadpoles can store their chemical defenses.

New paper in Zoomorphology

Slicing and Reassembling Toad Salami

It feels as if only a couple of months have passed since I last posted, although I can clearly see that it says Jan 22 there. Here is a quick recap of the last “few” months.

February to April I was focused on processing histological series of developmental serieshistology-photos of pairs of toad species that do and do not have complete tympanic middle ears. Our goal is to see how the development of head structures of eared and earless species is different. I was glued first to the microtome (think “salami slicer”), and then to the microscope (think “salami imager”). Right about the time that I had finished taking photos of each slice of each animal (~4000 photos like the one pictured), I learned that I had been awarded a postdoctoral fellowship from the American Association for University Women (AAUW; see profile here). The award is generally granted to later stage postdocs so that they can pay the bills while they analyze and write up data in the process of preparing for and securing a permanent research position. I am extremely grateful to have been awarded this honor and the timing could not have been more perfect!

Germany 2016 Devo poster.jpgIn May, senior team toad reps (Kim Hoke, Molly Womack, and myself) traveled to
Delmenhorst, Germany for a workshop titled “Linking Evolution and Development of the Auditory System” to present some posters on the toad project (mine is pictured to the right) and meet some fantastic colleagues studying similar questions. Who would have guessed there was such a specific conference just for the exact thing we are focused on!

Near the end of May and into late July, I shifted gears temporarily, and focused on coordinating the “fundamentals” graduate field course with the Organization for Tropical Studies (OTS) called “Tropical Biology: An Ecological Approach” along with co-coordinator Patricia Salerno, TA Victor Acosta, and a slew of amazing, skilled, and enthusiastic visiting professors. This course has been running for over 60 years since the first round with Dan Janzen and Jay Savage, taking students to different ecosystems around Costa Rica in a sort of bootcamp for early grad students. We do “experiment interval training”; at each site students have to design a hypothesis, collect and analyze the data, and present it in oral and written form (~1 week per project), and then move locations and rinse and repeat. Some projects are done alongside a visiting professor, sometimes in groups, and sometimes individually. By the end, students are much more confident and capable researchers. We also put a heavy emphasis on training in science communication, and the (biology graduate) students produce popular science articles, podcasts, and short films about the work they are doing on the course (see course VIMEO page here). Here’s an example film (that still makes me tear up), made by a group of 4 of our students with expert coaching from film producers Morgan Heim and Amy Marquis:

Patricia and I will be teaching the course again next summer (May 22-July 2, 2017), so if you are, have, or know of an early grad student that would benefit from some rigorous practice in developing scientific questions and following through to project completion, please send them our way!

Since the course ended in July, I have been glued to the computer focused on 2 major projects that will be the products of my time as an AAUW fellow.

First, I have just finished up with reconstructing 3D models of the developmental series of toads using a free program called “imod” and those 4000 salami photos I took in the lab. Labmate Molly Womack has written quite a bit about this on her blog, because she has done this for lots of juvenile and adult toads and bestowed me with her 3D reconstruction know-how.

In the video below of the 3D reconstructed ear of an adult toad, you can find the blue annulus, yellow and flat tympanum (the ear bit that we can see from the outside), and turquoise columella, the three famous parts of the middle ear that seem to disappear in a number of toad species. The big round gold part is the inner ear. Green is the operculum, which also transmits low frequency vibrations via the pink opercularis muscle and down through the scapula to the foot.

When I am not tracing these structures in each piece of salami, I am synthesizing information about the developmental timing of these structures in an extensive literature review. Because we believe that shifts in developmental timing are likely responsible for the mysterious loss of ears over 10 different times in this family, having this background of when structures appear across many species will give context and a timeline for the rest of our research. The literature review stretches from papers published in the 1880s up to things coming out this month, combining super-old school morphological observations with cutting edge genomics perspectives on neural crest cell differentiation, and aims to provide some solid hypotheses for future testing. One thing I have learned is that the style of writing in scientific papers sure has changed since 1881 when W.K. Parker published “On the structure and development of the skull in the Batrachia” in Philosophical Transactions of the Royal Society of London:

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In the last couple of months I’ve also had the chance to present the toad project in invited talks at the III Amphibian and Reptile Conservation International Symposium in Costa Rica and in a departmental seminar at the Universidad de Los Andes in Bogotá, Colombia. Team toad will be presenting updates on the projects I’ve mentioned above as well as Molly’s latest findings at the Society for Integrative and Comparative Biology (SICB) annual meeting in New Orleans, LA at 2 and 2:30 pm on Jan 5th in room 210.

Andes, Buses, and Toads

If you had suggested 5 years ago that I would spend almost 2 months crossing the Andes 6 times and sit ~100 hours on buses with Amazonian toads at my feet, I would have looked at you like a crazy person. #strangethingsforscience

This field season, Nov 2015-Jan 2016, was epic for me, and not only in the melodramatic sense of the word that the cool kids are using these days. First, I need to mention how honored I was to work alongside so many truly wonderful people throughout the journey. I learned so much and saw dozens of species of frogs and snakes and lizards while searching for toads with world-class Peruvian herpetologists like Amanda Delgado, Peter Condori, and Juan Carlos Chaparro. I laughed at the great jokes and huge grins of outstanding local guides like Santos and Hector who searched abandoned mining camps and road ditches with us until 4:30 am in the pouring rain. And with the help and care of fellow Colorado State biologists Dusty Gannon and Claire Tortorelli, as well as CDS research station owners Florencia Trama and Federico Rizo-Patrón (and their toddler Lorenzo), I was able to work through a number of logistical challenges, do science, and feel at home through a holiday season away from home.

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On the first of 3 major phases of the field trip, I flew up to Cusco and joined Peter and Amanda. We drove up and then down a rather crazy highway towards Puerto Maldonado in search of an arboreal toad.

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We got lucky with a heavy downpour on our first night there, and in a few nights were successful in finding all of the frogs here that we hoped to find and a few that I joy-squeaked a little bit when we first spotted (Amereega, Phyllomedusa, Oreobates, Osteocephalus, etc. etc.). We also found 6 snake species (2 venomous), a salamander (Bolitoglossa with its little moustache), and an anole.

Unfortunately, a lot of our conversation and dismay here was focused on noting the rapid expansion of illegal gold mining in the area. This activity involves tearing out big patches of forest along streams, using heavy machinery to dig up the stream beds and mud, pouring toxic chemicals like mercury into the sludge to pull out the gold bits, and then leaving all of the destruction and contamination behind. Its actually unbelievable that we still were able to find so many herps here given the conditions. We had a couple of interactions with miners who, although generally friendly, were obviously nervous about why we were there each night, and had us on edge as well. Do me a favor, blog reader…please do not encourage this gold industry by buying things made with gold.

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With a couple boxes of the Rhinella toads we were looking for (with permits in hand to transport them), we headed back to Cusco. Then I took a bus with the toads to Lima, because the permit process for flying with live toads is arduous as well as risky because the police could decide at any moment to confiscate or quarantine them. Generally I have a pretty stable stomach for bus rides, but this was the ultimate nausea inducing experience and I’ll just say that in the future I will gladly pay the extra $50 to fly up to Cusco. Once in Lima, I met up with insect hobbyist Cristhian, who sold me 350 baby cockroaches to feed the toads, and sent the toads and their crunchy lunch to Oxapampa on a bus.

Phase 2 of the trip was a whole other level of adventure. Joined by Juan Carlos Chaparro, author of many articles describing toad species and their natural history and curator of herps at a museum in Cusco, we ventured to the area of Chachapoyas in the north of Peru. Aiming for a toad that only lives above 2700m, in an area where mining and other questionable land use activities are rampant, one of our biggest challenges was obtaining permission. We had the permit from SERFOR, the Peruvian government organization for wildlife research, but needed to connect with the mining company and campesino community that oversees one of these mountain peaks to get their approval to hike up there. Juanca, who has an ideal character for this sort of thing, made it happen in the community of Shipasbamba. They asked us to attend an evening meeting with the elders and explain what it was we wanted to do. Although they found it a little unusual that this blond foreigner really wanted a few of their toads, they were excited to hear some stories about the herps that are unique to their area and seemed driven to ensure our experience in their community was positive (including an impromptu dinner for us!). In the end we received a handwritten letter, signed by the elders, giving permission to hike up into the abandoned mining camps on their land and look for the prized toad.

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With permits in hand, we were joined by 3 local guides, packed up a few days worth of food, and set to climb the 1800m to where the toads live in a dense population at the top of the mountain. The photos below can do a better job of describing this lifetime experience…

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Once back down, the toads and I set off for 3 days and nights of bus travel from Chachapoyas to Chiclayo, Lima, and finally Oxapampa. Now in phase 3 of the trip, I was joined by Dusty and Claire (recently graduated CSU biology students), Flor and Fede (directors of CDS research station in Oxapampa), and Peter (the same Peter from Cusco!). Our goal was to take the toads collected in the north and south, as well as a couple of species from the area of Oxapampa, and get them to make babies so that we can study the evolutionary development of their ears (or lack thereof). The process of breeding frogs comes with a whole set of challenges, and had never been attempted for any of these species, so we have had to overcome a lot of hurdles in the effort related to thermal and humidity conditions, food sources, habitat preferences, hormonal priming, and more.

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Now, Flor and Peter continue to monitor and encourage breeding through this wet season. I was able to bring back a bunch of samples and combining those with others that have arrived from our collaborators in Ecuador, I’m now working with CSU undergrads Koedi and Duncan to histologically process them. We hope to develop an understanding of the developmental morphology of species with and without ears so that later the Hoke lab can explore the molecular processes behind those morphological differences.

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And of course, while all of the science was happening, no trip to Peru would be complete without some creature comforts and a truly amazing array of FOOD.

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Animal Behavior (BZ300) complete!

Much of May and June has been focused on teaching a summer 3-credit course at CSU in animal behavior. With 22 students in tow, we plowed through 15 chapters of Breed & Moore’s Animal Behavior, watched and discussed many hours of behavior videos and case studies, and practiced writing and editing mock NSF GRFP proposals and presenting journal articles. Many comments from students as well as some formative and pre/post assessment assure me that the students enjoyed their month of animal behavior and got a lot out of it.

“The videos and group activities were really engaging and helped a lot with not only understanding but applying concepts. This was probably the most engaging and participatory course I have taken at CSU. Thanks for a great class!”

In spare moments between classes, bioinformatics work on guppy SNPs has continued, and I am gearing up to dive into a closer look at the developmental morphology of ear structures in our toads in the coming months with 3D reconstructions and microCT work. As a participant in the NSF GAUSSI program at CSU (gaussi.colostate.edu), training continues on bioinformatics and statistics techniques to visualize and understand genomics and transcriptomics data. Also, smile-inducing emails have come regarding the acceptances of a manuscript at the Journal of Herpetology and a quick guide at Current Biology.

In case you are hoping for more beautiful pictures of Peru, you should stay tuned for December. Flor and Fede are working hard caring for the growing youngsters in the cloud forest of Oxapampa, and I’ll head down to join them for the start of the wet season to repeat the process with a few other species. Our collaborators in Ecuador also continue to rear cute toadlets, and I’ll make a stop there on my trip later this year to learn from the experts in tropical frog breeding.

A Few Months In

Time for a little update. Since the last post, I’ve moved to Fort Collins, started working on teaching and research projects, and been back down to Perú for a 2 week visit. In Perú, I first stopped in Cusco with Kim and we met with collaborators Juan Carlos and Amanda to talk about frogs and permits and drop off museum samples (and to sample some local flavors).

Museo de Pisco

2015-03-22 11.26.162015-03-22 11.26.37Then, with a quick stop in Lima to pick up some new insect cultures to feed various sizes of toad, I hopped on the bus to Oxapampa and met with Flor and Fede. We spent time working on permits, refining protocols and data sheets, and checking in on the animals. One of the tadpole clutches started to get their little front feet while I was there, which is always an exciting time.

We also spent many hours searching around for more frogs so that Molly could test their hearing when she returns later this year. But, it seems as if the dry(er) season had already started, so those hours spent in the day and late in to the night amounted to moving lots of leaves with lots of sticks rather than finding frogs. But, given the location, it could have been worse.

Hunk lab postdoc uniform

Hunk lab postdoc uniform

Aside from the trip to the field site, I have been adjusting to life in the US after many years in Costa Rica. Some things are taking a while to settle in, such as the lack of extravagant hellos and goodbyes and cheek kisses regardless of how long it was since you last saw a person, which when suddenly absent feels a bit like you are being smugged off on a regular basis. But generally the adjustment has been relatively easy because Fort Collins is a charming town full of mountain trails, great food (and breweries!), quirky little cafés, and truly friendly people.

Heading to lab dinner

Heading to lab dinner

I’ve spent most of my time in these first few months on 4 main things. First, I have spent way more time than I ever thought would be necessary completing orientations, trainings, and sorting out the logistics of life, transportation, and being legally licensed and employed with benefits. I’m glad this phase of bureaucracy and paperwork is finishing up now.

Second, I have been preparing to teach Animal Behavior (BZ300) at CSU this summer. While I’ve spent a good amount of time dissecting the textbook and writing lectures, a lot of the time has also been spent coming up with engaging activities and reading potential discussion papers so that the students will actually be doing scientific thinking during class and not mindlessly taking notes. They’ll be writing funding proposals (GRFPs), developing experiments and hypotheses in class, and presenting a short journal article while learning the basic concepts of the field of animal behavior. I am thrilled at the opportunity to teach my favorite subject, and grateful that I have the time to invest ahead of the course to make sure it is a good one.

Third, I have been in constant contact with the folks in Perú to move along the permit process, troubleshoot over obstacles with rearing frogs and tadpoles, and talk about plans in the coming couple of years. Flor sends lovely photo and video updates so that I can live vicariously.

Lastly, I have been working on a piece of another of Kim’s grants focused on the behavior of Trinidadian guppies. My role is to analyze some existing transcriptome data, specifically focusing on using bioinformatics tools to find SNPs (single nucleotide polymorphisms, or places that the genes differ between individuals). It involves a lot of trial-and-error code writing while logged on to the supercomputer in our collaborator’s lab in Florida. I am having way more fun with this computer-based work than I anticipated, which is great because the upcoming analysis of the development of ears or not-ears in toads will involve similar work pipelines. I often forget that this work has anything to do with a pretty fish, but I still squeaked a little bit when I saw these first little blue dashes indicating SNPs after weeks of writing code scripts:

IGV shows guppy SNPs!

IGV shows guppy SNPs!

In the coming months, I’ll keep working on the teaching, guppy SNP, and toad rearing projects, and I’ll start mentoring some students in the lab to get going on histology (specimen slicing) of the development of ear bits in the toad samples we already have from Ecuador. The Hoke lab will also be attending the SSAR meeting in Kansas and the Evo Devo meeting in San Francisco in July and August. As they say in Costa Rica, “solo bueno, mae”.