Polytene chromosomes

This past spring/summer I had the opportunity to give talks at both the Drosophila Research Conference in San Diego, CA and the Evolution Conference in Portland, OR about my research on reproductive isolation and sexual selection in Drosophila pseudoobscura. During the Drosophila conference I was able to meet Steve Schaeffer (Penn State) who invited me down to State College, PA to learn his technique for polytenes.

Dper Muller A 2007_04_04_[Converted]
Polytene preparation of the XL chromosome and how it corresponds to a linkage map of the genome
Polytene chromosomes are formed in the salivary gland of Drosophila larvae when cells undergo repeated rounds of DNA replication without mitosis creating large cells. The DNA of these cells can be stained to show specific banding patterns that can identify chromosomes, specific genomic regions, and inversion karyotypes. I had been wanting to learn how to prepare polytenes to help identify inversion status of my wild-collected D. pseudoobscura collections.

Steve was a great host and I had a fun visit working on getting nice preps throughout the day. Steve has perfected his technique through his career and it has enabled him to make key insights about local adaptation maintaining inversions across the range of D. pseudoobscura.  To be able to distinguish banding patterns the chromosomes cannot be flattened too much or they become distorted, which was quite difficult for me as a beginer. Below is a preparation Steven made that identified a line from Lamoille Canyon, NV that was polymorphic for a inversion.

Dean Castillo LC8 Chr3

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Dissertation Defended

I successfully defended my dissertation April 12, 2016. Right before my dissertation defense my second manuscript looking at sexual interactions and sexual selection using C. remanei (First Chapter of my dissertation) was accepted at Evolution.

I was awarded a NSF postdoctoral fellowship and a NIH NRSA fellowship which I accepted to work in the lab of Dan Barbash at Cornell University.

IMG_6751

Picture: The Moyle lab (and Darwin) post-defense

New paper out on sexual selection and reproductive isolation

My most recent paper on the evolution of pre-zygotic isolation due to divergence in sexual interactions and sexual selection has been published in Evolution.

This project was started in collaboration with Lynda Delph and Curt Lively as a my rotation project (that continued for another year) as a first year student. Melissa Burger was an honors student in the Delph lab whose thesis included work reported in this paper.

http://onlinelibrary.wiley.com/doi/10.1111/evo.12815/abstract

A related manuscript examining genetic variation in mating traits in the ancestral population is currently being written up

nematode mating

Chromosome mapping of GFP insertions in D. pseudoobscura

I think the number of visible markers in D. melanogaster is often under appreciated by fly researchers. It really only became an issue for me once I started stepping out of D. melanogaster and started looking for markers in D. simulans and more recently D. pseudoobscura.

The marker that I utilized in D. simulans was a GFP insertion (Castillo and Moyle 2014, creation of flies explained by Holtzman et al 2010) because it was dominant and could be seen in F1 hybrids.

For D. pseudoobscura there are also GFP insertion lines, but for my work with D. pseudoobscura I ideally wanted to complete sperm competition assays between wild type genotypes (and not a standard lab genotype). My solution was to introgress this GFP marker into several isofemale lines. Luckily D. pseudoobscura has ~6X the recombination rate of D. melanogaster, which would allow quick introgression. The down side is the large inversions on the 3rd chromosome.

I decided to map the GFP insertions using a strain that is marked with visible-recessive mutations on all of its major chromosomes. If the GFP did not co-segregate with any of the mutations then I would have determined which chromosome the insertion was on. Below I share some of the phenotypes I was scoring in the backcross generation:

The back cross individuals were all yellow (X chromsome) and segregated for gl (glass eye phenotype) and or (orange eye color) with an appreciable frequency of double mutants. They also segregating the GFP insertion. Starting in the upper left corner and moving clockwise we have gl/gl;+/+, then +/+;+/+, then +/+;or/or, and lastly gl/gl;or/or. In the second picture (same flies but they have moved around some) you can clearly see the GFP insert in the +/+;or/or fly and it is also visible in the wild-type fly (its even easier to view in the ocelli). This indicates that the GFP insertion is on the second chromosome as it is never found with the gl marker.

pseudo eye colors copy gfp copy

Graduate Recruitment links

I forgot to post this link a couple of weeks ago in time for IUs Graduate Recruitment Weekend. A crazy snow storm almost assured that all of the recruits would attend IU because their flights would be cancelled indefinitely.

I really liked the strategy/topics discussed in the post. One takeaway I got form the post (even if it was not explicitly stated) was to take ownership of your research and be proactive instead of being passive, which can be a difficult transition for some first year students depending on their undergrad institution.

https://winninggradschool.wordpress.com/2015/02/15/tips-for-grad-students-especially-first-years/

New Paper in Proc Roy Soc B

My first chapter from my dissertation has now been published online so I thought I would share some of the inspiration and back story behind the project

http://rspb.royalsocietypublishing.org/content/281/1797/20142050.abstract?sid=4c5d8eda-48d3-49cb-8bed-6c7aa0d3714a

When I was an undergrad I spent 1.5 years working in the lab of Andy Clark on a sperm competition experiment with his postdoc Clement Chow. This project focused on the contribution of a particular pair of genes (Sex Peptide and Sex Peptide Receptor) to the variation in sperm competition phenotypes with Drosophila melanogaster. Concurrently I was taking a course on Speciation with Rick Harrison and was thinking a lot about gamete precedence as a barrier to reproduction and was surprised that genes contributing to this barrier had not been identified.
The idea of trying to connect within species sperm competition to conspecific sperm precedence floated around for a while and I actually wrote my NSF Graduate Research Fellowship on this topic, but I was more focused on genetic correlation between the two phenotypes. When I got to the Moyle lab and decided to work on Drosophila this was the first experiment I started and Leonie and I decided to focus on candidate genes with known effects within D. melanogaster (ongoing dissertation research is looking more at genetic correlation between phenotypes in natural populations). Using these genes allowed us to design a simple experiment that had a straightforward contrast between the effect of each gene on within species sperm competition and conspecific sperm precedence

Lamoille Canyon NV

The new semester is about to begin and I am still processing flies that I collected in July. As I have written about in a previous post identifying species in the obscura group (and really any Drosophila group) is a time consuming process between dissections, test crosses, and microsatellite work. For my experiments I am trying to collect two allopatric (with respect to co-occurence with D. persimilis) and two sympatric populations of D. pseudoobscura. This summer I focused collecting in the Sierra Nevada and in Lamoille Canyon. I had never been into the Ruby Mountains before and it was a beautiful place to camp and collect. After a short recon trip (and finding out there were no bears) I went for a longer four day trip living out of the back of my Dad’s pickup truck. Lamoille Canyon was carved by glaciers during the last ice age and is truly an island in the middle of high desert. Sage desert quickly transitions to mixed oak/poplar forest and then treeline in a 30 minute drive. The first generation from my isofemale collections will eclose this week

OLYMPUS DIGITAL CAMERA

OLYMPUS DIGITAL CAMERA

Latest Hadena-Silene publication

The last publication resulting from my work on Hadena-Silene interactions was finally published. I had a great two summers collecting data with my co-authors and learned so much about the writing process working on these manuscripts. Links to all three are below:

Castillo et al 2013 Specialist pollinating seed predator exhibits oviposition strategy consistent with optimal oviposition theory

http://onlinelibrary.wiley.com/doi/10.1111/een.12003/full

Castillo et al 2014 Invasive Silene latifolia may benefit from a pollinating seed predator, Hadena ectypa, in North America

http://www.jstor.org/discover/10.1086/673536?uid=3739664&uid=2&uid=4&uid=3739256&sid=21104424107703

Kula et al 2014 Interactions between a pollinating seed predator and its host plant: the role of environmental context within a population

http://onlinelibrary.wiley.com/doi/10.1002/ece3.1134/full

 

Evolution 2014 and CA trip

Now that the semester is over I am focusing my energy on writing/data analysis, before ramping up new projects.

I am also excited for the Evolution meeting this year.

http://evolution2014.org/

For various circumstances I have never made it to this meeting, and it will be my first national meeting since 2008.

After a quick turnaround I am making another collecting trip to CA and NV for Drosophila pseudoobscura and persimilis.