BEACON Researchers at Work: Evolution Goes Plink

Posted on November 16, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by MSU graduate student Kyle Card.

I visited the Pictured Rocks National Lakeshore over this past summer. Here I am sitting near Miners Castle looking toward Lake Superior.

I visited the Pictured Rocks National Lakeshore over this past summer. Here I am sitting near Miners Castle looking toward Lake Superior.

Richard Feynman was an eccentric theoretical physicist and Nobel laureate who had a profound impact on the field of quantum mechanics. As a child, he would take long weekend walks with his father in the woods. During these walks, his father taught him about the workings of nature and the importance of observation. He learned that it was all right not to know the answer to something. Rather, the journey to find the answer is fulfilling and exciting in its own right.

A classmate, who knew of these walks, approached Feynman in the schoolyard one day and pointed to a bird sitting on a distant fence. He asked smugly, “What is the name of that bird?” After Feynman’s puzzled silence the boy chided in, “It’s a brown throated thrush. Your father doesn’t teach you anything!”

Feynman would tell this story to stress a lesson his father had taught him: it doesn’t matter what the bird is called. One can learn the name of a bird in all the languages of the world and still know absolutely nothing about the bird. There is a fundamental difference between knowing the name of something, and knowing something. The observation of the bird’s behavior is what counts. Science is about coming to a deeper understanding of nature. We are constantly pushing outward the boundary between what is known and what isn’t, which often leads to more unanswered questions to explore. This has led some to liken nature to an onion: when its layers are peeled back, one often uncovers more layers. Or as Philippe Verdoux put it, “Enlightenment leads to benightedness; Science entails nescience.” Ultimately though, the scientist takes pleasure in the simple act of discovery itself.

Like Feynman, I have always been deeply curious about nature. However, for most of my childhood I was fascinated by the cosmos. At the age of seven I was reading about astronomy and trying to wrap my head around nuclear fusion. By the age of thirteen I awaited the release of Stephen Hawking’s book The Universe in a Nutshell like it was the next Harry Potter installment (I also grew up reading the Harry Potter series, so I was a somewhat normal child, I swear!). I remember the librarian remarking how the book may be too advanced for me and I should consider looking for another book. I didn’t, and well, she was right – sort of. It was advanced, but why should I have let that deter me? It wasn’t until high school that I became interested in biology, and not until half way through my undergraduate studies that I became fascinated with the living world that can only be seen through a microscope.

I am a microbiologist who learned about evolution on my own to better understand my undergraduate research. I find evolution beautiful and elegant, and I am often taken aback by its immense explanatory power. I have become particularly interested in how genes interact with other genes in the same genetic background, a phenomenon called epistasis, and how chance historical events can constrain evolutionary outcomes. To introduce these, I invite the reader to imagine the game Plinko from The Price is Right.

The Plinko board, in all its glory.

The Plinko board, in all its glory.

For those unfamiliar with the game: the Plinko board is very large and sits upright at a slight angle. Many small pegs protrude from its surface and at the bottom of the board lies nine containers. Each container has a corresponding prize amount. A lucky contestant climbs to the top of the board where he or she drops chips, one at a time, onto the pegs underneath. As the chip falls down the board it strikes the pegs, one after another, until it lands into one of the containers at the bottom. Whichever container the chip lands in, the contestant wins that amount. The best outcome for the player is to have the chips fall into the center container, which has the highest payout. The path a chip takes to the bottom varies every time the game is played. When the chip hits a peg, it may go left or right. The next peg it hits is dependent upon the direction it took during the preceding event. This process occurs all the way down the board until the chip lands in a container. If one were to imagine this occurring many times, each time a chip is released, it will take a slightly different path toward the bottom, and which container it falls into will likely differ between games. Thus, the outcome (reward) is dependent upon all preceding events that came before, along with the initial starting position of the chip itself. The outcome is further constrained by the limited number of containers; “the paths are many, but the destinations are few,” according to Simon Conway Morris.

Stephen Jay Gould proposed that a similar path dependence, which he termed historical contingency, largely makes evolution unpredictable. If one were to “rewind and replay the tape of life” multiple times, different evolutionary outcomes will emerge based on the sequence of historical chance events that occur. Gould’s proposal has been controversial, and only empirical investigation of it will determine if it is correct.

Historical contingency may play an important role in antibiotic resistance. Antibiotic resistance is a serious and pervasive threat to healthcare. According to the CDC, two million people will acquire antibiotic resistant infections every year in the United States alone. Among those two million, 20,000 will die. Antibiotic resistance is a product of natural selection in pathogen populations, and therefore, it is important to study this process from an evolutionary perspective. History may be important, because when a resistance mutation is introduced into a given genetic background, either through chromosomal mutation or on a plasmid, the level of resistance and fitness cost of the mutation are determined in part by the mutation’s interactions with other genes in that background. And the background itself is the product of a long history of chance evolutionary events, much like the Plinko example above. I am studying historical contingency’s role in antibiotic resistance using the long-term evolution experiment (LTEE) with Escherichia coli in the lab of Dr. Richard Lenski.

The LTEE consists of twelve E. coli populations that were founded from a single common ancestor and have been evolving for 63,500 generations, or over 27 years. Thus, it offers a unique opportunity to study the impact of history on evolution, as has been demonstrated by the work done on the evolution of aerobic citrate usage in one population. In the case of antibiotic resistance, the same resistance mutation may have different levels and costs of resistance in different LTEE populations because they have different genetic backgrounds due to their different evolutionary histories during the experiment.

Look mom, I’m doing science!

Look mom, I’m doing science!

As a new graduate student in the lab, I am still in experimental “dry dock.” In the coming months, I aim to test the level and cost of resistance of multiple antibiotic resistance mutations in the ancestor and clones isolated from all populations in the LTEE at 60,000 generations. I will test level of resistance using minimum inhibitory concentration assays. (This is just a fancy way of saying I will introduce increasing levels of antibiotics to my bacteria until they die. Then I will note how much antibiotic it took to kill them. I’m sorry little guys, but it’s for science!). To measure any fitness costs of resistance mutations, I will run pairwise competition assays comparing resistant mutants to their sensitive kin without the antibiotic present.

The observation of different levels and costs of resistance among multiple LTEE populations would suggest that historical contingency is likely playing a role in antibiotic resistance. Moreover, studying antibiotic resistance as an outcome dependent upon antecedent events is important to understanding its role in diverse medically relevant microbes with varied evolutionary histories.

Separate from this project’s medical relevance, I find the questions driving the research deeply interesting. For as long as I can remember I have been keenly fascinated with history. As a kid growing up, I watched the History channel quite a bit (before its sad downturn into Ancient Aliens and Pawn Stars), as well as The Price is Right (as any sick child staying home from school can attest). So maybe it was inevitable I would find a way to infuse both into my research, at least to some degree.

For more information about Kyle’s work, you can contact him at cardkyle at msu.edu

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BEACON Researchers at Work: Searching for the Cradle of Life…

Posted on November 9, 2015 by Danielle Whittaker

This week’s blog post is by MSU faculty member Matt Schrenk (Dept. of Geological Sciences & Dept. of Microbiology and Molecular Genetics).

IMAX flange on the northwestern side of the 60-meter-tall Poseidon chimney at the Lost City Hydrothermal Field, venting 55°C, pH 11 fluids. Image courtesy of IFE, URI-JAO, Lost City Science Party, and NOAA.

IMAX flange on the northwestern side of the 60-meter-tall Poseidon chimney at the Lost City Hydrothermal Field, venting 55°C, pH 11 fluids. Image courtesy of IFE, URI-JAO, Lost City Science Party, and NOAA.

… a kilometer beneath the sea. We set sail from Southampton, UK in late October aboard the RRS James Cook on IODP Expedition 357 (X357). Our mission: to drill into the Atlantis Massif, a 5 km tall mountain along the Mid-Atlantic Ridge, and home to the Lost City hydrothermal field- a series of ethereal-looking chimney structures which many believe are an analog to where life on Earth first originated. Earlier work at Lost City found that circulating hydrothermal fluids contain energy and organic compounds, usually considered to be the product of life, originating from abiological processes deep within the Earth. Microbial communities use these compounds- primarily methane and hydrogen, to fuel their metabolism and form prolific biofilms within the chimney structures. But, on this trip, we hope to get beneath the chimney structures- to the ‘root zone’ where these primitive biochemical reactions occur, and to see what life, if any, survives there. One of the primary goals of our expedition is to identify how microorganisms in the subsurface use these compounds, and what this can tell us about the emergence and evolution of life on early Earth and perhaps other planets and moons.

An array of diamond impregnated drill bits used on the bottom-hole assembly to drill into rocks of varying hardness at the bottom of the seafloor. Core barrel is 61 mm across. Image courtesy of Y. Morono (JAMSTEC).

An array of diamond impregnated drill bits used on the bottom-hole assembly to drill into rocks of varying hardness at the bottom of the seafloor. Core barrel is 61 mm across. Image courtesy of Y. Morono (JAMSTEC).

We set off on X357 as a team of microbiologists, petrologists, organic geochemists, and petrophysicists with two seabed rock drills to access the fractures and pore spaces of the sub-seafloor. During our long transit to the Lost City at 30°N and 42°W (or the middle-of-nowhere to anyone looking at a map), we went over and over and over again our procedures for sampling the rock cleanly- for keeping the carbon and organisms and DNA from ‘surface’ life from contaminating our precious samples. The level of detail in planning has been extraordinary, from the delivery of ultra-sensitive PFC tracers into the drilling fluids, to the attempts to track and constrain the origin of carbon (especially surface-derived ‘living’ carbon) on the ship, to taking dozens and dozens of samples from every conceivable spot- even the greases the driller’s use!- to source track microbial DNA. Perhaps the most extraordinary part of the project are the seabed rock drills themselves, which robotically bore through solid rock a kilometer beneath the sea surface, operated remotely by technicians onboard the ship. On this cruise we have two of these drills, allowing us to go continuously for almost a month, alternating between the MeBo (Marum- University of Bremen) and the RD2 (British Geological Survey).

I have worked with samples from the Lost City before, having spent the better part of my doctorate squeezing DNA out of rocks, and trying unsuccessfully to domesticate the microbes found within chimney materials. As my Ph.D. advisor would probably say- the ‘holy grail’ would be if we could obtain an organism from Lost City in pure culture, to study if these organisms really are ‘living fossils’, and if their genomes and their biochemistry tells us something about the earliest biochemical pathways. I spent months prior to the cruise preparing hundreds of tubes of different growth media to cast a wide net to capture this ‘organism X’, guided by clues of what we have learned over the last decade, and hopefully some intuition. My fingers are crossed. Fortunately, in the time since my graduate work, a far more reliable approach has emerged through the high throughput sequencing of DNA. Even if our cultivation attempts are not completely successful- we can still use approaches such as shotgun metagenomics and single cell sorting and sequencing to reconstruct microbial genomes and to conduct evolutionary comparisons to other organisms and genes. Hence the comprehensive tests we are doing for contamination and context.

Photo of Rock Drill 2 (RD2), owned by the British Geological Survey, being deployed at 30°N near the Mid-Atlantic Ridge. The drill holds a carrousel of core barrels that allow penetration to a depth of up to 50 m below the seafloor. Image courtesy of Y. Morono (JAMSTEC).

Photo of Rock Drill 2 (RD2), owned by the British Geological Survey, being deployed at 30°N near the Mid-Atlantic Ridge. The drill holds a carrousel of core barrels that allow penetration to a depth of up
to 50 m below the seafloor. Image courtesy of Y. Morono (JAMSTEC).

The Lost City system is fascinating from an early evolution perspective. Rocks originating in the Earth’s mantle are brought to the surface by plate tectonics and react with water through a process known as serpentinization, liberating carbon from the deep Earth, and catalyzing the formation of methane and other short-chain hydrocarbons. These processes were likely even more common early in Earth’s history, before the planetary crust had completely distilled into the different materials we have today. Hydrothermal circulation moves seawater through the fractures and pore spaces of the subseafloor- across this catalytic mineral matrix, before emerging at the seabed. The circulation sets up gradients between hot, highly alkaline (pH 10+) fluids and seawater, which some have argued resemble early cell membranes and set up natural proton gradients which are a common feature of all cells on the modern Earth. Within this catalytic milieu, ‘single-species’ biofilms of methane-cycling Archaea grow tightly bound to the mineral surfaces, duplicating genes and physiologically diversifying to an unprecedented degree.

At least, that is what we think we know- the new samples will undoubtedly introduce new wrinkles in the story. It will be difficult to detangle 3.8 billion years of evolutionary history by pulling on only one string. Nevertheless, it is exciting to have the chance to look at these vaults of biodiversity hidden deep within our planet and hear the stories that they might tell.

As I write, the RD2 drill is making slow but steady progress with its diamond drill bit eating through meters of solid rock. First cores should be on deck within the next few days. The entire ship is poised and anxious to turn our best-laid plans into action. Hopefully, we will soon meet this proto-organism that lives beneath the seafloor.

For more information about the expedition, see: http://www.edo.ecord.org/expeditions/357/357.php

or

www.schrenklab.com

 

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BEACON Researchers at Work: Omics beyond model organisms, part II

Posted on November 2, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by MSU postdoc Gaurav Moghe.

Almost two and half years ago, when I was a graduate student, I wrote a blog post in this very series titled “Omics beyond model organisms”, wherein I attempted to make a case that developments in omics technologies offer a fantastic opportunity to explore biology in non-m

odel organisms, of which a vast majority are still largely unstudied. Almost 2.5 years later, I find myself as a postdoc in the thick of this very exact research, working on understanding how the massive diversity in specialized metabolism (or secondary metabolism as is popularly known) arises in the plant world.

How did I end up doing this research? While I was working towards my PhD, I also created a wiki website called Biodiversity of India on the side, which documents various aspects of Indian biodiversity. I’m originally from India, where I was brought up with various Hindu religious customs, traditions and folk medicines that had significant associations with the local flora and fauna. I was interested in documenting such cultural associations of biodiversity, in hopes of not only aiding biodiversity conservation efforts but also organizing the information to make it more accessible for research. While working on this site, I became interested in plant specialized metabolism, which constitutes the basis for several of our spices, folk medicines, perfumes, cosmetics and pest control agents.

Word cloud of different types of plant specialized metabolites. Size of words is scaled by the log10 of the approx. number of known compounds (Wink, 2010).

Word cloud of different types of plant specialized metabolites. Size of words is scaled by the log10 of the approx. number of known compounds (Wink, 2010).

There are >200,000 specialized metabolites known in the plant world, and they come in various different shapes and forms. These compounds are produced by plants for interacting with the living world around them – their pollinators, microbial communities, herbivores and other plants around them. Thus, specialized metabolites constitute a highly diverse “language of the plant world”. However, the various evolutionary mechanisms that generate the observed natural variation in this language are not well-documented. Imagine a world where we KNOW the language of plants! Not only will that give us a profound appreciation of the innovations in the living world, it will also provide us an opportunity to harness those innovations for societal uses such as in agriculture or medicine.

I joined Rob Last’s lab here at MSU to work on the evolution of specialized metabolic pathways in the plant world, specifically on a class of compounds called acylsugars, in tomato and related species in the nightshade (Solanaceae) family. These sticky compounds are produced by hairy tissues called trichomes on the surface of leaves and stems, and they are important for insect defense. Acylsugars were known to be present only in some Solanaceae species, but no one had yet conducted a broad analysis of acylsugar phenotypic variation in Solanaceae. In addition, I was interested in understanding how the biosynthetic pathway was put together – did it exist when the family arose? When did the enzymes arise? What did they emerge from? How have the enzymes evolved since their emergence? The process of addressing these questions would also unravel diverse biosynthetic enzymes that may be used in a “toolkit” for acylsugar production in cultivated tomato, thus boosting its natural defense against pests.

To address these questions, I performed high-throughput mass spectrometric analysis of over 40 Solanaceae species, catalogued the extensive acylsugar phenotypic variation (>200 different acylsugars documented) and selected four species with very interesting phenotypes. Then, I performed RNA-seq from the trichome and non-trichome tissues of these non-model species and identified candidate genes using differential expression and orthology. Finally, using a combination of in vitro enzyme assays and in vivo gene knockdown strategies, I was able to confirm some of the enzymatic activities that produce the observed phenotypes in different Solanaceous species.

Homology model of an acylsugar biosynthetic enzyme, with positions of the two substrates modeled into its active sites. Rapid evolution in the binding pockets may lead to rapid divergence in enzyme activities.

Homology model of an acylsugar biosynthetic enzyme, with positions of the two substrates modeled into its active sites. Rapid evolution in the binding pockets may lead to rapid divergence in enzyme activities.

My results indicate that the acylsugar biosynthetic enzymes – that belong to an enzyme family known as the BAHD family – may have emerged from alkaloid biosynthetic members of the same family, and the activities may have existed close to the emergence of the Solanaceae. My findings also suggest that these enzymes are highly evolvable and rapidly change their substrate and donor specificities, and that frequent duplications and subsequent functional divergence may have led to the observed diversity of acylsugars we see in the Solanaceae family today.

Several questions still remain unanswered. For instance, what causes the rapid divergence of activities? Are the diverse acylsugar phenotypes functionally important, or is a higher order trait such as “stickiness” more important? Are the enzyme activities evolving via drift, or are certain lineages under selection? Are the BAHD enzymes under selection to be more evolvable? The original question – what did these enzymes evolve from? – also remains unanswered. My future research is aimed at addressing some of these questions.

Overall, I feel that my PhD and postdoctoral research have given me a strong foothold in using biodiversity-driven, experimental as well as computational approaches to understand plant physiology. This strategy is enabled by the rapid development in omics and phenotyping technologies. In the future, I hope to create platforms where we can conduct such research in non-model plants and enable rapid discovery of evolutionary processes in the plant world.

For more information about Gaurav’s work, you can contact him at moghegau at msu dot edu.

 

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BEACON Researchers at Work: The grasshopper mouse versus venom

Posted on October 26, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by MSU graduate student Abhijna Parigi.

Abhijna ParigiOf all the bizarre animals that live in the deserts, grasshopper mice are, objectively, the cutest. These cinnamon-colored rodents are small enough to fit in the palm of your hand, weighing no more than a common house mouse. In the hot, dry deserts of the southwestern United States and northern Mexico, grasshopper mice have adopted a nocturnal lifestyle, where they use their large black eyes and highly sensitive ears to navigate in the dark. Unlike most rodents, grasshopper mice are vicious carnivores that regularly hunt reptiles, other rodents, and large arthropods. In the dead of the night, they are known to stand up on their hind legs and howl at the moon, before using their quick reflexes and sharp teeth to hunt prey. However, the wolf-like habits of grasshopper mice are not their most striking traits.

Grasshopper mouse hunting a scorpion. Photo by Dr. Matt Rowe

Grasshopper mouse hunting a scorpion. Photo by Dr. Matt Rowe

All three known species of grasshopper mice (genus Onychomys) share their habitats with, and regularly hunt, a number of venomous and toxic arthropods. One, the bark scorpion, uses painful neurotoxins both as a defense against its predators and as a means of capturing and paralyzing its prey. Over evolutionary time, in response to the strong natural selection imposed by bark scorpion envenomation, grasshopper mice have evolved physiological resistance to bark scorpion toxins (A. H. Rowe & M. P. Rowe 2008). The amount of scorpion venom that can quickly paralyze and kill a common house mouse seems to have little effect on the grasshopper mouse. The Rowe lab at Michigan State University focuses on understanding how: 1) grasshopper mouse nervous systems have evolved in response to selection by pain-inducing venoms, and 2) how venoms themselves have changed over time. We think that grasshopper mice and bark scorpions may be involved in an escalating evolutionary arms race, where toxic scorpion venom selects for more resistant mice, which in turn select for more toxic scorpions.

Mommy bark scorpion (Centruroides sp.) with her babies. Photo by Josh Goldston

Mommy bark scorpion (Centruroides sp.) with her babies. Photo by Josh Goldston

Pain signals are detected and transported to the brain via the action of three types of specialized proteins called voltage-gated sodium ion channels (Nav1.7, Nav1.8 and Nav1.9). Because the ability to sense pain is crucial for survival, functional sodium ion channels are highly adaptive, and their structure is conserved across many diverse taxa. In other words, even small changes (mutations) to these proteins may dramatically alter the electrical properties of neurons, and manifest as neurological disorders. For example, in humans, mutations in the Nav1.7 gene are shown to cause many pain disorders ranging from complete pain insensitivity to the sensation of intense chronic pain.

Desert giant centipede (Scolopendra heros) in captivity. Photo by Josh Goldston

Desert giant centipede (Scolopendra heros) in captivity. Photo by Josh Goldston

Previous work in our lab has shown that grasshopper mice have evolved a clever way to (at least in part) shut down pain signals induced by bark scorpion venom, without compromising their ability to sense other types of pain. In grasshopper mice, sodium channel Nav1.8 carries structural modifications that allow the channel to bind with venom and block the pain signals that the venom was originally meant to induce (Rowe et al. 2013). That is, the venom acts as its own painkiller. Although the role of grasshopper mouse Nav1.8 in scorpion venom resistance was an exciting discovery, it is only a small part of much larger story. Grasshopper mice feed on a variety of other neurotoxic prey, including tarantulas and centipedes, which defend themselves by using sharp mandibles to inject their predators with painful venom. As we know nothing about how grasshopper mice react to tarantula and centipede venom, I am studying the effects of tarantula and centipede venoms on the electrical properties of different sodium channel isoforms involved in the pain pathway. In particular, all three venoms (centipede, tarantula and scorpion toxins) contain peptides that bind to Nav1.7, a channel that is critical for pain sensation (Dib-Hajj et al. 2012). An additional fact that makes Nav1.7 interesting is that this channel is also expressed in one other tissue – the olfactory epithelium, where it plays a vital role in mediating the sense of smell (Weiss et al. 2011). This dual functionality of Nav1.7 makes it especially suitable for the study of evolutionary trade-offs. Evolutionary changes to the functional properties of Nav1.7 that alter pain sensitivity may inadvertently change the channel’s functionality in the olfactory tissue – perhaps compromising the animal’s sense of smell. On this front, an exciting result has been the discovery that the grasshopper mouse Nav1.7 gene (as expressed in pain sensing neurons) carries structural modifications. Further, these modifications are located in areas of the channel that may be involved in mediating its electrical properties.

Grasshopper mouse poking its head out of its laboratory home.

Grasshopper mouse poking its head out of its laboratory home.

My greatest challenge in the Rowe lab thus far has been to clone the Nav1.7 gene for expression in a commercially available mammalian cell line. Doing so will enable me to use electrophysiological techniques to compare the electrical properties of the modified grasshopper mouse Nav1.7 channel to that of the wild-type house mouse Nav1.7 channels. To get to the bottom of whether or not Nav1.7 substitutions in the grasshopper mouse pain-sensing neurons trade-off with the animal’s sense of smell, I am conducting behavioral olfactory sensitivity assays. These simple assays will test for differences in the threshold at which grasshopper mice and house mice can smell specific odor cues (e.g. peanut butter, urine, predator scent). Although I do not have any results to share at the moment, our preliminary data suggest some exciting patterns that we are eager to explore with further experimentation.

Organ Mountains: one of our field sites.

Organ Mountains: one of our field sites.

References:

Dib-Hajj, S.D. et al., 2012. The Nav1.7 sodium channel: from molecule to man. Nature Reviews Neuroscience, 14(1), pp.49-62.

Rowe, A.H. & Rowe, M.P., 2008. Physiological resistance of grasshopper mice (Onychomys spp.) to Arizona bark scorpion (Centruroides exilicauda) venom. Toxicon, 52(5), pp.597–605.

Rowe, A.H. et al., 2013. Voltage-gated sodium channel in grasshopper mice defends against bark scorpion toxin. Science, 342(6157), pp.441–446.

Weiss, J. et al., 2011. Loss-of-function mutations in sodium channel Nav1.7 cause anosmia. Nature, 472(7342), pp.186–190.

For more information about Abhijna’s work, you can contact her at parigiab at msu dot edu.

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BEACON Researchers at Work: A foreigner’s forays into experimental evolution

Posted on October 19, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by MSU graduate student Carina Baskett.

In my native habitat, looking for caterpillars on pokeweed plants.

In my native habitat, looking for caterpillars on pokeweed plants.

When I spent a semester of college in Buenos Aires, Argentina, we American students were endlessly fascinated by cultural differences between the US and Argentina. As we awkwardly learned to navigate the warm Argentine culture (e.g., it is customary to kiss everyone in the room upon arriving a party), we found it symbolic that there is no Spanish translation to describe someone as “awkward.” The rules of the road were also notably different, put best by an Argentine who had recently visited the US. “It’s so weird!” she told her friends. “People actually use turn signals when they’re changing lanes!”

I found myself in a foreign land again last winter and spring, when I was working on an experimental evolution project with bacteria and viruses at a lab bench. You see, I am a plant biologist, and most of my data comes from fieldwork. I am happiest when sitting in front of a flower and watching for pollinators to alight, or when turning over a couple thousand leaves to find a few caterpillars. Plants fascinate me; although lacking brains or the ability to move, they have adapted creative ways to have sex, disperse their seeds, defend themselves from hungry hungry caterpillars, and survive winter.

So how did this plant geek end up working on an experimental evolution project? I asked myself that question many times, especially when things weren’t working! The short answer is that even though plants are awesome, they are too big and grow too slowly to directly address the question I was burning to answer.

I talked in detail about that question here: why are the tropics so diverse? Although I do get to work with plants in nature for part of my research, another part of it is only possible by conducting an evolution experiment with bacteria. I will save the details of our study for a future post, when I can talk about results. Right now, it’s still very much a work in progress.

Designing the project involved many hours of brainstorming over breakfast and beer (usually not at the same time) with my collaborators, Alita Burmeister, Luis Zaman, and Justin Meyer, not to mention sage advice from Ben Kerr and my advisor, Doug Schemske. We came up with some ideas about how to recapitulate some essential differences between tropical and temperate environments in the lab, and obtained BEACON funding to study how those different environments would affect evolution of bacterial diversity.

Alita and I worked together last winter to get some preliminary data for her research while I learned the ropes (the flasks?) of microbiology research. That was when the language barrier really hit me! Don’t tell my collaborators, but I can never remember which important genes and proteins are which in our system, despite their patient, repeated explanations. Somehow the function of “ompF” just doesn’t stick in my head as well as the image of “Carolina horsenettle.”

Despite my poor memory for these details, I learned a ton from working with Alita in preparation for the next step. In early March, on a grey day that I think was the first above freezing in at least a month, I flew to sunny San Diego to spend three months conducting our carefully designed experiment in Justin’s brand-new lab at UCSD, where Luis was setting up essential new equipment for the project.

That equipment was a device called a “morbidostat,” which would allow us to use antibiotics to control bacterial death rates, even as they evolved resistance. It sounds great, but it’s a new device, whose technical issues are still being worked out—by us! It didn’t take long before we realized that a lot of our carefully designed ideas and plans weren’t going to work exactly as we’d hoped. This was a familiar realization from my fieldwork experience, but I wasn’t expecting to encounter it in the lab. I thought we could control everything perfectly in the lab! I thought we could get beautiful data by starting a bacterial culture, pressing some buttons and typing programs into a computer!

The flask on the left is cloudy with a large E. coli population, despite the presence of a virus. The population in the flask on the right has crashed, likely because the virus has evolved a new way of invading the bacterial cells. How cool is that??

The flask on the left is cloudy with a large E. coli population, despite the presence of a virus. The population in the flask on the right has crashed, likely because the virus has evolved a new way of invading the bacterial cells. How cool is that??

Turns out that biology, chemistry, and luck—nature, in other words—can wreak havoc on the best-laid plans of microbiologists, not just field ecologists. For instance, I spend several weeks of my limited time there battling a mysterious, recurring precipitation in the media that we were using to grow our bacteria. Eventually I found that we had been adjusting the pH higher than we should have been, an issue that was exacerbated to the point of precipitation because the pH meter had become slightly off-kilter…possibly my fault, for not storing it properly over one night.

I know this kind of setback is completely normal in all aspects of science, but in those frustrating moments I wondered whether I preferred the headaches from fieldwork to those from the lab. It’s hard to decide though, because the rewards from experimental evolution are great. I still have photos of flasks and plates on my phone from the experiment that Alita and I worked on almost a year ago, and I still pull them out to show people how amazing it is to be able to see evolution happening from one day to the next. For example, we would come into the lab sometimes and see that a flask that should have been cloudy with bacteria was clear, an indication that overnight its virus had evolved a new way to bypass bacterial resistance. Evolution in action is really exciting! Even this plant geek can freely admit that.

So will I pursue dual-citizenship in the worlds of plant field ecology and microbial experimental evolution? I’m not quite sure yet. When (not if!) we eventually finish this project, I’ll decide whether I enjoy the work enough to look for a post-doc in an experimental evolution lab. If so, I hope that someday working in the lab won’t feel so awkward.

A virus entering a bacterial cell, or a scarlet macaw flying out of a tree? Hmmm...

A virus entering a bacterial cell, or a scarlet macaw flying out of a tree? Hmmm…

For more information about Carina’s work, you can contact her at baskettc at msu dot edu.

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BEACON Researchers at Work: Are Electric Fish Magic?

Posted on October 12, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by postdoc Will Pitchers from the Gallant Lab at MSU.

Will PitchersThe fishes in our lab — African freshwater fish called Mormyrids — seem pretty magical when one first hears about them; they spend their lives surrounded by a pulsed electric field they generate themselves, and consequently perceive the world, in some very unintuitive ways, with (proportionately) one of the largest brains of any fish. As they can sense their surroundings electrically they can navigate and hunt in complete darkness, or the murkiest of waters. The output of their electric organs also allows them to communicate with each other over a ‘private channel’ on which their prey (mostly freshwater invertebrates) and predators cannot eavesdrop. None of this is the sense of ‘magical’ that I’m referring to however; I’m talking about ‘magic traits’ as coined by a Prof. called Sergey Gavrilets (2004) at U. of Tennessee.

To explain: we know that sometimes speciation can occur in sympatry — i.e. a species splits in two in situ — but it can be hard to find a mechanism for how this happens. What could keep groups separated long enough to evolve apart while they share a habitat? It’s not hard to imagine fishes from one population specializing on one of a couple of food sources, e.g. some rootling for buried worms while others glean for insect larvae. However, this kind of niche partitioning won’t led to speciation as long as the two groups interbreed. In order to split the species, there also needs to be some reason for the two groups to breed assortatively; preferring mates from their own group rather than the other. A so-called ‘magic trait’ would be one whose evolution could begin to diverge for ecological reasons, but which could also lead to sexual incompatibility. Evolving ‘magic traits’ could therefore put species on the fast-track to speciation.

Electric fish specimensWe think that the electro-perception of Mormyrid fishes might fit this bill quite neatly; since they both hunt and court each other using their electrical pulses. Mormyrids’ electric organ discharges (EOD’s) differ from species to species, and there’s some evidence that these electrical properties influence what features of the fishes’ environment are perceived most clearly (Von der Emde 2006; 2010). It seems likely therefore, that selection could ‘tune’ the EOD to be efficiently detect a preferred food source. On the other hand, we know that Mormyrids identify which fish are the appropriate species with whom to mate by their electrical signals (Feulner et al. 2009) and evidence suggests that females are choosy on the basis of EOD (Schmid & Kramer 2014). The coincidence of the EOD’s importance in both foraging and mate choice might go some way towards explaining why the Mormyrids are so unusually diverse, having split into ~200 species over 20 genera.

Trying to get to the bottom of how the potentially ‘magic’ EOD might be influencing Mormyrid evolution is one of the main research thrusts in the Gallant Lab at present. For my part, I am using the computing resources at iCER to sift through a big heap of next-gen sequencing data to see if we can associate which bits of the genome are associated with variation in the properties of the EOD. As I’m a new recruit to the electric fish scene, I’m going to have to leave you on a cliffhanger, but watch this space for exciting discoveries as we make them!

Gallant lab members

References

Emde, Von der, G. 2006. Non-visual environmental imaging and object detection through active electrolocation in weakly electric fish. J. Comp. Physiol. A 192:601–612. Springer-Verlag.

Emde, von der. 2010. 3-Dimensional scene perception during active electrolocation in a weakly electric pulse fish. Front. Behav. Neurosci. 1–13.

Feulner, P. G. D., M. Plath, J. Engelmann, F. Kirschbaum, and R. Tiedemann. 2009. Electrifying love: electric fish use species-specific discharge for mate recognition. Biology Letters 5:225–228. The Royal Society.

Gavrilets, S. (2004). Fitness Landscapes and the Origin of Species. Princeton University Press

Schmid, D., and L. B. Kramer. 2014. Sexual selection by female choice prevents speciation reversal in a hybridizing trio of mormyrid fish in southern Africa: evidence from playback experiments of electric organ discharges. Behaviour 151:1703–1734.

For more information about Will’s work, you can contact him at pitchers at msu dot edu.

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BEACON Researchers at Work: Female vision-related genes are more plastic in Bicyclus anynana

Posted on October 5, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by University of California at Irvine graduate student Aide Macias-Muñoz.

Aide Macias-MuñozMy interest in biology began when I was very young with my fascination in living things. My exposure to diverse plants and animals mostly took place every summer when I traveled with my family to Nochistlan, Zac., Mexico. I was very interested in the care of domesticated farm animals and the large diversity of moths and butterflies that I saw in this small town and rural surroundings. I often overwhelmed my parents with questions that they did not have the answers to.

During my undergraduate career at UC Berkeley, I majored in Integrative Biology and took a broad range of courses. Through these courses I began to learn the answers to many of the questions that I had as a child, and, in the process new questions emerged. After joining Adriana Briscoe’s lab at UC Irvine, I acquired new skills in molecular biology and bioinformatics and began tackling these questions.

Aide Macias-Muñoz at microscopeOur lab uses different approaches to investigate the evolution of sensory systems in insects, primarily focusing on evolution of vision in butterflies. My role in the lab has been to explore gene expression by creating and analyzing RNA-Sequencing libraries. My most recent project sought to understand how gene expression of vision-related genes changes in a species that exhibits phenotypic plasticity for vision.

Bicyclus anynana is a butterfly species that has two seasonal forms corresponding to the wet and dry season. The distinct seasonal forms display an interesting behavior of sex role reversal. In the wet season, males court females, which choose mates based on UV-reflectance of the dorsal forewing eyespot pupils. However, in the dry season, females court males that now exhibit choosy behavior (Prudic et al. 2011). A previous study, showed that eye size varied between sexes and seasonal forms; males had larger eyes relative to females and wet season forms had larger eyes in both sexes relative to dry season forms. In addition, opsin expression complemented sex role reversal; long-wavelength, blue, and ultraviolet opsin genes had decreased expression in non-choosy dry season female butterflies (Everett et al. 2012).

Photo credit: Antónia Monteiro. Bicyclus anynana wet and dry season form mating. The wet season form has conspicuous eyespots and a pale band on its wings, while the dry season form has cryptic coloration and reduced eyespots.

Photo credit: Antónia Monteiro. Bicyclus anynana wet and dry season form mating. The wet season form has conspicuous eyespots and a pale band on its wings, while the dry season form has cryptic coloration and reduced eyespots.

In order to see what additional vision-related genes were associated with eye size differences, we did a differential expression analysis of sexes and seasonal forms. We found that non-choosy dry season females do indeed down-regulate blue and ultraviolet opsin mRNA relative to wet season females. We also identified two eye development genes and an eye pigment biosynthesis gene differentially expressed between seasonal forms. Upon closer inspection, we found that the biggest magnitude change was in dry season females.

Our results show that differential expression of eye development and eye pigment genes is associated with divergent eye phenotypes in B. anynana seasonal forms, and that there is a larger effect of season on female vision-related genes. This difference is potentially due to variation in energetic demands between the sexes. Females have the additional metabolic burden of producing eggs and thus may be under greater selective pressure to reduce non-essential physiological functions (Macias-Muñoz et al. In Press).

References:

Prudic KL, Jeon C, Cao H, Monteiro A. 2011. Developmental plasticity in sexual roles of butterfly species drives mutual sexual ornamentation. Science 331:73–75.

Everett A, Tong X, Briscoe AD, Monteiro A. 2012. Phenotypic plasticity in opsin expression in a butterfly compound eye complements sex role reversal. BMC Evol Biol. 12:232.

Macias-Muñoz A, Smith G, Monteiro A, Briscoe AD. Transcriptome-wide differential gene expression in Bicyclus anynana butterflies: Female vision-related genes are more plastic. MBE. In Press. doi: 10.1093/molbev/msv197

For more information about Aide’s work, you can contact her at amaciasm at uci dot edu.

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BEACON Researchers at Work: Life by the Lake

Posted on September 28, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by College of Charleston junior Kola George. Kola was an BEACON Undergraduate Research Apprentice (URA) at MSU Kellogg Biological Station in summer 2015, with Jeff Conner as his mentor.

Kola_friendsThis summer, I was fortunate to participate in research under the direction of Dr. Jeff Conner concerning evolutionary trait loss. Along with my research, I had many great experiences all of which contributed to an amazing summer filled with science, new experiences, and new friends. Growing up in a rural area, I was always outside playing in the woods or embracing what nature has to offer. Because of this, I was excited to spend time taking advantage of the breathtaking landscape of the Kellogg Biological Station.

Kola_labFor my research project I tested whether the number of short stamens in the model plant Arabidopsis thaliana, which is a highly-selfing plant, impacts the number of seeds produced. A previous study by Dr. Anne Royer showed that A. thaliana appear to be losing their short stamens and she suggested the short stames had lost most, or all, of their function. To examine this idea, I was able to work with 10 populations from across Europe from Belgium, Germany, France, Czech Republic, Spain, Sweden and Italy. We grew 2 plants from each population and I counted the stamens daily without harming the flowers, and marked each flower individually. After two weeks, I counted all the seeds in the fruits that were produced on each plant. Data from our experiment suggested that short stamen number did not affect seed set, and thus the short stamens do appear to have lost their function!

KBS Gull LakeIn addition to an engaging research project, there were many other things going on at the Biological Station, all of which made this a great summer. I camped on the shores of Lake Michigan, hiked the dunes at Sleeping Bear National Dunes National Lakeshore, and spent an entire day exploring the Field Museum in the city of Chicago. Living at KBS on the shores of Gull Lake the whole summer was also an amazing thing in itself. Throughout the summer I got to get to know a lot of cool people and played hours of soccer, basketball, and volleyball to unwind from a long day in the lab. There were days that I felt homesick, but the constant energy that came from Gull Lake and my friends at KBS made it so I could never have a bad day at KBS! From the many experiences I’ve had at KBS, one of my favorites has to be ending most of my days watching the sunset at the dock on Gull Lake. It never failed that every time I watched the sun set it was more of a sight to see than the time before. This summer has provided me with life-long friends, professional development, a great research experience, appreciation of nature and the desire to know more about the world that surrounds us.

 

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BEACON Researchers at Work: Murphy's Law

Posted on September 21, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by MSU sophomore Krista Nicholson. Krista was an BEACON Undergraduate Research Apprentice (URA) at MSU Kellogg Biological Station in summer 2015, with mentors Susan Magnoli & Dr. Jen Lau.

My partridge peas in the greenhouse stubbornly not blooming.

My partridge peas in the greenhouse stubbornly not blooming.

Murphy’s Law states that what can go wrong will, and there may be no better field of science to apply this to than evolutionary ecology. I started my Undergraduate Research Apprenticeship (URA) at Kellogg Biological Station with the illusion that every experiment would work, and that I would always get satisfying results. I didn’t know any better, every lab I’d ever done in school had turned out pretty much exactly like the directions said they would. I soon found out that natural systems don’t like to follow directions.

My main task as a URA was to help my mentor Susan Magnoli with her experiments this summer. She studies how genetic diversity may be beneficial in prairie restorations, so we worked in a handful of prairies close to KBS. For the first half of the summer we set up plots in two of those prairies, cleared them of debris, planted thousands of partridge pea plants from six source populations, and watered all of them. Then all of our plants were eaten by grasshoppers and voles.

Lauren and Aaron my busy helper bees.

Lauren and Aaron, my busy helper bees.

After the devastating blow dealt to us by the voles and grasshoppers, all we could really do was use the remaining plants in the greenhouse to set up a few smaller experiments. Susan gave me a number of those plants to use in my own pollinator experiment. Time passed, my plants got bigger, and soon it was the last few weeks of my internship. Despite getting big and healthy, most of my plants never blossomed.

By this point I was losing hope of collecting my own data. There didn’t seem to be any flowering partridge peas that I could use! Mark Hammond, the lab tech for the Lau lab, heard of my plight and came to my rescue. He offered the use of some partridge peas in heating rings at the Long-Term Ecological Research site as long as I helped him collect some data he needed. I was elated, finally a chance for me to do my own science! I decided to examine the impact of ants and rhizobia on pollinator visitation in partridge peas grown 3ºC above ambient temperatures. My mentor and I were at the site by 9 am with our comfy lounge chairs and a coffee and set ourselves up to watch the pollinators stream in. Almost none came.

A bumblebee pollinating a partridge pea.

A bumblebee pollinating a partridge pea.

Again I was nearly ready to give up. I had my plants, they were blooming, but I couldn’t force the pollinators to visit! Then one of the people working in the rings earlier in the morning mentioned to my mentor that he saw pollinators around 8 am, and that if we got there earlier we might see more. Susan and I got up bright and early the next morning, I brought an even bigger cup of coffee, and we set up around 7:30 am. Lo and behold, there were dozens of pollinators! We had difficulty even keeping track of all of them. I was ecstatic, and every day for the next week after we had collected a new set of data I would go back to my room and excitedly input it into excel.

Soon the symposium was approaching, and Susan and I (mostly Susan) analyzed the data. A lot of coding in R and some graphs later, I had my results; the manipulated variables had no direct effect on pollinator visitation. The only significance we found was that plants with more flowers attracted more pollinators, and that plants with rhizobia had more flowers. This wasn’t a direct effect, and because of this I was disappointed. How was I supposed to make a poster and present on an experiment without strong results?

It was then I realized something. Real research isn’t perfect. I had been told all summer that most experiments fail, but I didn’t realize that applied to me as well. It would have been more surprising if I had found something significant actually! In fact, my observations and results inspired me to come up with a new experiment that I hope to conduct next summer. It finally hit me that if I wanted to do research and be a good scientist I would have to be okay with frequent failure. All most people see is the final product, the glossy paper published in some eminent journal. But real science is about getting your hands (and everything else) dirty, it’s about getting up early, staying out late, failing, redesigning your experiment and failing again, it’s about overcoming obstacles and finding novel solutions, and most of all it is about never giving up. I also realized then that I wouldn’t have it any other way.

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BEACON Researchers at Work: EDAMAME!

Posted on September 14, 2015 by Danielle Whittaker

This week’s BEACON Researchers at Work blog post is by MSU faculty member Ashley Shade.

We received overwhelmingly positive feedback from our Explorations in Data Analysis for Metagenomic Advances in Microbial Ecology (EDAMAME) workshop last year, which was partially supported by BEACON and MSU’s iCER. It was immediately obvious that EDAMAME was addressing an urgent unmet need in the scientific community. The data from our course evaluation (thank you, BEACON, for facilitating our interactions with STEM ED, LLC to evaluate our workshop!) showed that EDAMAME learners had achieved our overarching goals of increased confidence and competence in microbial sequencing data analysis. In short, we seemed to be doing a lot right and having a positive impact on our learners’ abilities to own their microbial sequencing analyses. We were ecstatic.

EDAMAME Group photo credit: Tom Rayner, @tomonlocation

EDAMAME Group photo credit: Tom Rayner, @tomonlocation

This year, we had double the number of applicants from last year, and many of them were so exceptional that distinguishing among them for the purposes of admissions was challenging. We have reached out to recruit a broader applicant pool than last year, and I see the benefits of our efforts reflected in the diversity of backgrounds and academic interests represented among our EDAMAME learners this year. We also had a portion of our applicant pool from governmental organizations like the USGS and EPA, and also from not-for-profit organizations, which reflects a need beyond academia for the flavor of training that we provide. In retrospect, the high level of interest from many different universities, institutions and agencies makes sense: microbes are the functional foundations for all ecosystems, from human bodies to soils to deep-sea vents. Why shouldn’t there be wide interest in learning how to observe and analyze Earth’s ubiquitous but functionally elusive microbial communities?

At EDAMAME, we strive to provide the best learning materials and instruction that we can, and there is always room for improvement! For #edamame2015, we’ve refined our learning objectives (listed at the bottom of this post) and backwards-designed tutorials to meet those objectives. We’ve also spent more time on topics we glossed over last year like starting, using, and transferring files to an Amazon EC2 instance, which we hope will help learners who do not have access to a high performance computing cluster to know how to access the computing resources needed to execute analysis of our ever-larger sequencing datasets. After feedback for “MORE TIME” from last year’s EDAMAME learners, we expanded to 10 days so that we can spend more time with the more complex material. We also scheduled time for independent study with instructors available to give learners the opportunity to analyze their own datasets with our support. We additionally organized our tutorials in a GitHub wiki (with a CC-BY license) so that folks outside of the course can more easily find and use our materials. Help yourself!

TAs: Siobhan, Jackson, Paul, Sang-Hoon, and Jin

TAs: Siobhan, Jackson, Paul, Sang-Hoon, and Jin

And, this year, with almost a full year behind me on the tenure-track at Michigan State University, I also was able to bring with me my own new team of students and post-docs to serve as teaching assistants for the course. They bring contagious enthusiasm, patience, and experience to the course (they took EDAMAME in its inaugural year in 2014, even before joining my team). At every break, we collect “minute cards” (borrowed form Software Carpentry best practices) to receive immediate feedback from learners on what is going well and what needs to be addressed. With this feedback, our learners “spoke” loudly: our TAs were just amazing. I am so lucky to be supported by these stellar young scientists, including: Siobhan Cusack, Dr. Sang-Hoon Lee, and Jackson Sorensen from my group; Paul Wilburn from Elena Litchman’s group at Kellogg Biological Station; Dr. Jinlyung Choi from Adina Howe’s group at Iowa; and Aaron Garoutte from Jim Tiedje’s group at MSU.

We also recruited a celebrity line-up of local microbial ecology geniuses as guest speakers, including MSU’s Jim Tiedje, Matt Scholz, as well as RDP’s Jim Cole and Qiong Wang; KBS’s Sarah Evans and adjunct Ariane Peralta (East Carolina University); University of Michigan’s Vince Young, Vincent Denef, and members of the Schloss research team; Indiana University’s Jay Lennon; and the University of Notre Dame’s Stuart Jones.

Within a week and a half, our learners dive in to an array of computational and bioinformatics topics. We covered navigating the shell, cloud computing, remote sessions for running long jobs, within-sample and comparative diversity, merging paired end MiSeq reads and QIIME and mother for microbial amplicon analysis, shotgun metagenome analysis (assessing quality, digital normalization, assembly, annotation), R for ecological statistics, RDP tools and their new exciting targeted gene assembler Xander, using high performance computing resources, and accessing public databases. During breaks, there was volleyball and campfires and the backdrop of the summery Kellogg Biological Station on Gull Lake.

We were thankful and excited to be awarded transition funds from BEACON’s internal small grants competition to support our workshop this year until we found external funding. We couldn’t have continued the workshop without BEACON’s interim support. And… an announcement! I am pleased to share that the National Institutes of Health have taken an interest in EDAMAME, and we just have been awarded EDAMAME support for an additional three years! So, EDAMAME onward!

Thank you, again, BEACON for supporting EDAMAME!

Ashley Shade @ashley17061

Assistant Professor, Microbiology and Molecular Genetics

Newbie BEACON member – since 2014!

EDAMAME 2015 Learning Goals

  1. Increase computing literacy
  2. Develop proficiency in cloud computing
  3. Analyze microbial amplicon sequences
  4. Analyze microbial shotgun metagenome sequences
  5. Apply ecological statistics to analyze and interpret microbial sequencing data
  6. Access resources provided by public sequence databases

 

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