**This post is a part of the series on the 2019 AGA Presidential Symposium – Sex and Asex: the genetics of complex life cycles**
About the author: Kara M. Million is a PhD candidate in the Lively lab at Indiana University. Her dissertation work is on parasites, mate choice, and MHC gene diversity in Darters (colorful little freshwater fishes). Kara enjoys hiking, drawing comics, and spending time with her husband and pets (a black cat and a python).
“Remember: flow cytometers can smell fear.”
Christiane Hassel, the manager of our university’s flow cytometry core facility, told me with a wry grin in the 8-week graduate course on flow cytometry I’d signed up for to round our credit hours for my PhD.
I wondered whether I was getting more than I bargained for. The confirmation came when Christiane casually mentioned that each student needed to design and execute a final project for the course.
I felt the familiar tide of panic rise within me. This was it. This was how everyone was going to find out that I was an impostor who didn’t belong here. I was a second-year PhD student who had moved to Indiana from Huntsville, Alabamafor grad school. Curt Lively, a world-renowned evolutionary biologist, had recruited me to join his lab. The lab’s flagship study system is a species of New Zealand snails (Potamopyrgus antipodarum, “potamos” for short) that have co-occurring sexual and asexual populations in the wild. Potamos are a powerful model for evaluating the evolution of sex. So why on earth did Curt hire a fish biologist?
I don’t belong here,I thought as I stood for the first time before the massive LSR-II flow cytometry machine, a behemoth affectionately nicknamed “Hades”. Christiane glanced at Hades with pride as she explained how it worked. A flow cytometer runs tiny particles, such as cells, through a fluid stream one at a time. Lasers strike the particles, which have been labeled with fluorescent dyes, and the machine uses the resulting fluorescence to measure something about the particles: size, count, cell cycle stage, nuclear DNA content… the possibilities are virtually endless. The Lively lab had a long tradition of using flow cytometry to tell sexual (diploid) snails from asexual (polyploid) snails based on nuclear DNA content. But I didn’t work on the snail system. What was I going to do for my project?
I don’t belong here, I thought again as I muddled through the first few training sessions on the flow cytometers. I made so many mistakes! What I didn’t realize was that Christiane had seen it all and was virtually unflappable. She was kind, knowledgeable, and endlessly patient with me as I slowly grasped the principles and skills I needed to “run flow”. Under her guidance, I improved over time until I could run an experiment on my own with minimal errors. I was proud of my newly acquired superpowers, but I still had a problem: what was I going to do for my final project?
I swallowed my fear and pride and approached Curt in his office. Sunlight streamed over his desk, which was dotted with papers and coffee cups.
“I like your office,” I commented, “It’s so nice in here. Lots of natural light.”
“I like it too,” Curt said cheerfully, “Hard to believe I started out in the basement.”
“You had your office in the basement?” I said with astonishment.
“Oh yes,” he said, “I had to work my way up to this room.” His eyes twinkled with merriment.
“I need a project for my flow cytometry class,” I said, “And I… I have no idea what to do.” More like I have no idea what I’m doing, I thought.
“Not a problem!” said Curt, “I have just the thing…”
Curt explained that the Lively lab had founded a bunch of clonal snail lab lines. Each line had been isolated from a single asexual female, and most of them had been maintained in the lab for years. The lab had also founded and maintained several sexual lines. Some of our collaborators had observed high variation in nuclear DNA content in field populations of these snails. Not only did sexuals and asexuals differ in their ploidy levels, but there was fine-scale variation between individuals in exactly how much DNA was in their cell nuclei. Curt wanted someone to measure DNA content within and between our clonal lineages in the lab. This was my mission should I choose to accept it. Two clones from the same line could appear to be genetically identical and have the same ploidy, but would they have the same DNA content as well?
Zoe Dinges and Amrita Bhattacharya, two other Lively lab grad students, oriented me to the “wet lab” and presented me with the lab’s official flow cytometry protocol. The protocol had been passed down from labbie to labbie, with each user contributing occasional tweaks. As I ground tissue, mixed reagents, and filtered cells through fine mesh, I felt as if I were participating in a sacred ritual. I felt connected to my fellow lab members and to the others who had occupied this space before me.
The LSR-II was equipped with a High Throughput Sampler (HTS), which allowed me to run an entire 96-well plate of samples in one go. The flow cytometer hummed and grunted as it picked up each sample, ran it through the stream, and hit it with lasers. The samples were stained with propidium iodide, an intercalating agent that would bind to the DNA in the cell nuclei and allow the machine to measure DNA content based on fluorescence. The higher the fluorescence intensity peak, the higher the DNA content in the sample. Using a trout erythrocyte standard with a known DNA content, I calculated mean DNA content for each sample.
To my astonishment, I observed levels of variation beyond my expectations. Not only did mean DNA content differ substantially between clone lines, but there was high DNA content variation within each line! Even more fascinating were the samples in one line that showed double fluorescence peaks, indicating possible ploidy mosaicism! These observations raised another question: how did the variation in DNA content compare to the variation in nucleotide diversity within and between these lineages?
To answer that question, I teamed up with Amrita, who was a veteran in the snail system doing molecular work on the snails as part of her own research. Together we piloted and perfected the methods to extract high-quality genomic DNA samples from the snails, a challenging task due to the high mucus content in the sample tissues. We recruited two plucky undergrad researchers in the lab, Eries Smith and Sarah Montgomery, to help us process our samples. In no time at all the two had become an unstoppable duo in the wet lab preparing pristine DNA samples for genotyping. Amrita and I beamed at each other with pride as we watched our mentees laughing together at the bench, their gloved hands and pipets working in almost perfect synchrony. The future of science rested in their skilled hands and their bright, joyful minds.
We obtained SNP primers from Gerlien Verhaegen, a colleague in Germany, and sent our samples away to be genotyped at dozens of sites across the genome to evaluate nucleotide diversity within and between our snails. One day while working in the lab we got that first fateful email: “Your genotyping results are ready.”
Curt walked in right as Amrita and I received the news. “Curt! Curt!” we shouted almost in unison as we jumped up and down, “We did it! It worked! We have DATA!”
The genotyping results revealed that snails within a clone line had identical genotypes across all sites, but that there was considerable variation between the lines. We expanded our genotyping study to include samples Curt and Zoe had collected from the field in New Zealand. Zoe had been toiling in the field, at the bench, and in the flow facility for her dissertation research. The field samples were taken from the same lake (Lake Alexandrina) from which the lab line founders had been collected years ago. We were amazed to discover little to no overlap in genotypes between our field samples and our lab lines. Was natural selection driving a high rate of clonal turnover in the lake? Curious-er and curious-er!
Maurine Neiman, another collaborator and former Lively lab member, suggested that we test whether DNA content correlated with SNP diversity in our snails. A Mantel test revealed no correlation, suggesting that these characteristics were evolving independently of each other! Meanwhile, Amrita had performed a series of parasite exposure experiments on our lab lines and had unearthed variation in susceptibility between our lines. What had started out as a small project for an 8-week graduate course had ballooned into a massive, multi-year research effort. As we analyzed our data and attempted to form and write conclusions, we became overwhelmed. There were so many moving parts, so many findings to communicate. What story were we trying to tell, and how were we to tell it?
Our rescue came via Zoe. Besides her deep intuitive grasp of the lab’s main study system, Zoe had acquired impressive coding and data visualization skills. It was Zoe who translated our raw findings into beautiful (and readable!) figures. By the time we submitted our manuscript to Journal of Heredity, we had become a close-knit team. Zoe, Amrita and I were leading the charge as the primary authors. Sarah and Eries were powerhouses at the bench and enthusiastic participants in the manuscript preparation and revision process. Curt, as always, was our anchor, keeping us conceptually grounded and providing invaluable guidance (not to mention funding!).
By the time our paper was published this winter, Amrita had moved on to a postdoc, our undergraduates had graduated, and the remaining lab members found ourselves separated due to a global pandemic. Even though we were far apart physically, the project had kept us all tied together in spirit. When the paper was published online, we celebrated our success through virtual means.
I’m still a fish biologist. Curt let me build my own tiny lab nested inside the Lively lab. I’m currently conducting my dissertation research on parasites, mate choice, and immunogenetic diversity in colorful little fishes called darters. However, this snail project helped me find my unique place in the Lively lab and on Team Potamo. I can finally say with confidence: I belong here.