I *heart* Data

PhD comics never fails to have an appropriate image. (http://www.phdcomics.com/store/mojostore.php?_=view&ProductID=12631)

 A while back I said I would discuss what “comes next” after all the field work. Well, I have started the next big project: identifying all the ticks I collected from the mice and voles we trapped over the summer. This is a big project because I collected over 670 ticks, and these are larvae and nymphs, the younger life-stages of the tick, and are really small and can only be identified under a dissecting microscope. It is important to identify all these ticks to species and life-stage because there are different assumptions about previous host interactions and possible infections for each group. To catch everyone up, here is some background before we get too deep into things.

This is a diagram of the Ixodes scapularis lifecycle. It is a "cool season" tick because its adults are active in the fall. Most of the ticks in Indaina have all life-stages active in the spring and summer.

To review, ticks are ectoparasites of vertebrates that have a 4-stage life cycle: egg, larva, nymph, and adult. These parasites need to bite a host and obtain a blood meal in order to molt and transition to the next life-stage. This is somewhat unique because other common vectors like fleas and mosquitoes only feed on a host directly in the adult stage. This means ticks can have many interactions with host over their lifetime and have the opportunity to become infected or pass on infection many times. The larvae emerge from eggs uninfected, besides obligate bacterial symbionts, so the larva can pick up a pathogen infection during their first blood meal. When these larvae molt into nymphs they become infected with whatever bacteria was picked up during the previous blood meal and can transmit to an uninfected host. This same thing can happen with adult ticks, but they have had two possible times to pick up an infection. The adult female ticks then feed to produce eggs, the males mate with the females during this blood meal and they rarely feed themselves. Then the females drop of the host and lay thousands of eggs in a “mass”. The larvae then emerge in the spring and the cycle starts all over again.

Mouse with a lot of engorged nymphs on its back.

The rodent hosts that I am interested can be hosts to the larval and nymphal stages. In the part of southern Indiana where I conducted these surveys have three main species of tick, but only two have been found using rodents as hosts, Ixodes scapularis (blacklegged deer tick) and Dermacentor variabilis (American dog tick). The main character that differentiates larvae from nymphs is that larvae have 6 legs while nymphs have 8 legs (as well as the adults). Each species has some unique features that I use to discern between the two.

My lab bench

The tools I use when identifying ticks are a dissecting microscope, this is a microscope that doesn’t need a specimen to be prepared on a slide. It used an external light source to illuminate a whole sample. I manipulate the ticks mainly with a paintbrush. This is a common tool for people who handle fragile invertebrates in the lab because it can move and stick to the specimen but won’t accidentally damage it like a normal pair of forceps. There are also “soft” forceps that are made from a flexible metal that are useful for samples that are too heavy for the paintbrush to grab on to. I covered my whole bench in white bench paper. This helps keep my workspace clean, and if any ticks fall or get dropped they will show up better on the light background. My scope to chair hight ratio is still off a bit, the chair is too high so I have to hunch to look into the dissecting scope, which can be a little painful after a long time. I’m going to have to figure out the best solution to this problem (a shorter chair or raising up the scope on a platform or something).

Dermacentor is the most common tick we’ve found on the rodents, from our previous surveys and from the data I’ve collected from this past year so far. Its mouthparts are somewhat rounded, the body and legs are a light brown color, and the shield on the back (dorsal) side comes away from the body in a straight line.

Ixodes seems to be somewhat less common on these hosts, but still very present. This is of particular interest to many because this species can carry Borrelia burgdorferi, the bacteria that causes Lyme disease. These ticks have longer, straighter mouthparts, are usually darker, blacker in color (the legs in particular, especially in the larvae), and the shield is rounded all the way around. The picture I have below compares a nymph from each species. See if you can see some of the differences I listed.

These little rodents are kind of teeming with parasites, and ticks are the only ectoparasites I collected from them. Fleas and mites were fairly common, but are harder to collect because they don’t attach to the host so they are freely moving through the host’s hair and around the body while you are trying to grab them with forceps. These parasites are important because they can also carry pathogens that can infect wildlife and humans (remember theplague post?). Analyzing the blood samples we collected in 2009 showed that many hosts are infected with Bartonella, a flea-borne pathogen, so fleas may be really important in this disease community.

Flea from a mouse, most likely Orchopeas leucopus.

Fleas and mites look really different from ticks and each other. I know fewer details about these creatures, but I do know they look pretty nasty. Fleas are covered in these little hairs which make them really sticky to the host fur (and hard to handle with the paintbrush, my usual tool for this). They look like they're "swimming" through the hair when you see them on a host, its pretty crazy.

I couldn't get the whole mite in focus at once. The left picture shows its arms in focus, while the right picture shows the little hairs that cover its back.

I think mites look like little monsters with their front set of legs reaching above their head. I’m sure many of you think these guys all look like monsters, but I get so used to looking at ticks I’m not phased by them anymore (what a strange state to be in, huh?).

If I haven't completely grossed you out with this post, stay tuned for more on parasites!

The in-between time, or the bliss of not having students for a little while

http://www.phdcomics.com/comics/archive.php?comicid=1458

Welcome to 2012, everyone!

Sorry I’ve been so remiss on writing the last few months, the fall semester kind of got away from me with teaching and everything else on my plate. Hence why I am writing now, when a lot of people are still on vacation, and the subject of today’s musings, what this between semester time is like.

Like I’ve mentioned before, life as a PhD student means you are almost always working on multiple things, all that should get your full attention but never do. During the semester for most of us the main things on the plate are teaching and research. Teaching pays the bills and the research is what we’re actually here for, writing a thesis to earn our degree. Our teaching obligations always seem manageable at the beginning of the semester, but invariably end up taking up far more time than we expected, eating into our time and brain power for working on our research.

This fall I would have spurts of tons of grading to work on, which I would alternate with spurts of working on a manuscript, getting a fellowship application submitted, or getting lab work done. The few times when I had a whole week or two without grading were amazing, I felt really productive, I got to really think about my science, and got a lot accomplished without having the nagging feeling that I should be working on teaching in the back of my mind. I had this same feeling a couple days after we submitted our final grades when I had uninterrupted hours to really focus on getting a near final version of a manuscript edited. I got in this great zone where things seem to make sense that didn’t before and the quality of the work was a lot higher than it had been.

When you’re an undergrad, you take your last final and then are excited to get to be on vacation, do nothing or whatever you want for a few weeks. As a grad student, you are really excited to get some time sans teaching to get some of your own important work done. This was the first year, I think probably ever, that I wasn’t ready for Christmas when the semester was over. I wanted more quiet limbo time before the holidays to get to work on what I needed to work on, not what I had to work on for teaching, meetings, other semester-related obligations.

I love this graph. I think the garland along the best-fit line is my favorite part (R^2 value is 0.69, for anyone who is interested, hehe) (http://www.nature.com/nature/journal/v450/n7173/full/4501156a.html)

The holidays did come, but I didn’t really feel like I was on vacation until the afternoon Dec. 23^rd when I sent of a manuscript draft to a collaborator in Israel (they don’t have Christmas break there, hehe). I saw a small article in Nature a few years ago discussing how scientists aren’t giving themselves enough down-time, showing that the number of papers submitted on Christmas day has been increasing (see above image). While I do think expectations for working on weekends and holidays can be a bit high, I have a new perspective on this pattern. I think submitting a paper on Christmas, or at least over the holidays, would be kind of a good idea. There wouldn’t be any scheduled meetings, classes, or students wanting to come meet with you. You have some quite down time to gather your thoughts, and then maybe nip down to present opening or Christmas dinner after your work is done. When its work on your own time it is much more enjoyable, even if it is during a holiday.

Please don’t get me wrong, I really enjoy teaching and I like interacting with students. But as I’m discovering, as a senior PhD student there is always something to do, work that needs to be done and that you actually want to do sometimes, and this makes it hard to enjoy your other professional obligations that that time away from those things. Can you put “full-time multi-tasker” on your CV?

I hope to make time for blogging this semester. I have a lot of lab work and things on my plate, so I might try writing some more detailed posts about what my research consists of these days. Good luck in the New Year!

P.S. I wanted to write about a story from This American Life that came out in the middle of November, but clearly I didn’t have time. I would highly recommend listening to the first story about the collaboration between a cancer scientist and a music professor and about how scientists and non-scientists think very differently about the same problem. If people would like to discuss it I would be happy to have this as the subject of a future post!

http://www.thisamericanlife.org/radio-archives/episode/450/so-crazy-it-just-might-work

Bring out your dead! (so their plague genome can be analyzed)

Many of us may have had our first introduction to medieval history thought this great Monty Python movie. And as is their nature, this bit from “The Holy Grail” is pretty accurate. The Black Death, the epidemic of plague that ripped through medieval England and Europe, killed thousands of people and led to creation of the middle class (people were finally in demand once 30-60% of them died). But I digress. The reason I’m writing about the Black Death is because a paper came out last week in the journal Nature describing a new genomic analysis of bacterial DNA samples collected from infected people buried in a cemetery outside of London. There was a great summary paper published in the same issue that described what the current paper found and some of the controversies that exist between scientists in this field. I highly recommend reading this, I think it is very widely accessible. Below I discuss some of the things I learned from the papers and why I think it’s interesting. (Note: unless you have access through a university you may not be able to see the primary source paper. Hopefully everyone can get to the summary paper, though)

First, a little background. Bubonic plague is caused by a flea-vectored bacteria, Yersinia pestis. Human cases of the plague are rare but still occur (are easily treatable with antibiotics) but plague epidemics are a big problem in some wildlife populations, such as prairie dogs in the American west. The Black Death describes the plague epidemic that occurred in Europe between 1347-1351. Since the sanitation conditions in a big city like London was probably more abysmal that we can imagine today, you can expect there to be fleas everywhere. So, the main questions people studying these old strains of the bacteria are trying to answer are, Why did the Black Death kill so many people?, and Why are cases today much less severe than symptoms described during this ancient period?

Two hypotheses that tried to explain these observed differences between the 14^th century plague and cases today are that the Black Death wasn’t actually caused by Yersinia and that a more virulent strain of the bacteria was the culprit for the deaths during medieval times. The first of these hypotheses was rejected when Y. pestis bacteria was recovered from the teeth of two individuals known to have died of the plague during the 14^th century. However, some researchers weren’t convinced of this conclusion because it was very difficult to repeat these methods, i.e. it is really had to get usable DNA from human remains that are 600 + years old and make sure the results aren’t contaminated. Subsequently, if it is hard to recover samples that are in good enough condition to be analyzed, it is almost impossible, with the tools available in the early 2000s, to construct a genome with a good amount of confidence, so scientists were not able to test that second hypothesis.

Enter this new paper and new fancy technology! Researchers from Canada, Germany, and the United States began collaborating to unearth new information from bodies buried in the East Smithfield cemetery (or “plague pit” as some called it) that was used during the Black Death. Samples that had been collected and stored over many years were re-analyzed using what is called next-generation pyroseqencing. This is a really amazing method that amplifies DNA from a sample thousands of times with extreme precision. This means that even if there is a very small amount of bacterial DNA in a tissue sample, it will be amplified to the point of detection. Older methods, like PCR (polymerase chain reaction), which is still the workhorse of a lot of microbiology, were not sensitive enough to extract detailed DNA sequence information from these medieval tissue samples. So, this new analysis was able to compile this new sequence data into a genome of Yersinia pestis and compare it to modern samples of this bacteria and see how they are related.

What they found was that the ancient and modern strains of the bacteria are very similar, showing not a whole lot of evolutionary changes between the 14^th century and now. It also showed that the Black Death strain of Yersinia is probably the ancestor to modern strains, meaning that the Black Death strain of the bacteria is the source of most modern cases (see the paper for a picture of the phylogeny if you’re interested). This means that the “strain with increased virulence” hypothesis that was proposed is not supported by this data. What this suggested to researchers was that there was some kind of environmental interaction with the Yersinia infection that caused people to be much more susceptible or have more severe symptoms. This could be due to wars that were happing around this time, wearing down the men that came back from fighting. Also, there are other flea hosts besides the rats living in the city, which may change the probably transmission of the bacteria to humans if the fleas were infected from this alternate reservoir.

These findings are interesting for a lot of reasons, but some are very close to my research. First, I am planning on using the sequencing method used by the authors to analyze the bacterial communities in my ticks and rodent blood samples. Analyzing “meta-communities” is one of the main applications of next-generation sequencing because it gives you so much data on the composition, in terms of number and quantity, of microbial communities that wouldn’t be possible with previously used methods. Second, the finding that the ancient and modern strains of the bacteria are very closely related highlights how complex the pathogen-host interaction is. How a host will respond to an infection depends on a whole suite of characteristics, of both host and pathogen. Scaling up from the individual, how a population will respond to an epidemic will depend greatly on population composition, taking into account age structure, host quality or health, contact rates, and immuneocompetence. Some of these aspects may be very difficult to measure when your population lived over 600 years ago, but I bet the researchers in this field have methods they can use to figure them out. It is quite amazing what we are able to do now to investigate questions that have been unanswered for so long. And since this is a hot topic, I am sure there will be competing genomes and other analyses published in the near future.

I haven’t watched “Monty Pyton and the Holy Grail” for a long time, but I have the feeling I’ll be bringing this paper up next time. Sorry in advance to any family or friends that will have to bear with my extreme nerdyness!

A parasite more disgusting than ticks

** Warning: This post is going to maybe too gross for some people, so if you really are not too cool with parasites you may just want to skip this one (hehe).

The little white-footed mice that are the focal host of most of my research aren’t just hosts for ticks. They are the habitat for a multitude of macro- and microparasites. For instance, in our research we have collected three kinds of ectoparasites, ticks, fleas and mites. Many other researchers have studied intestinal worms, both acquired trophically (through eating infected food, like crickets), and by simple contact with worm eggs in the habitat. They can carry many vector-borne pathogens, bacteria and other microbes carried by ticks and fleas, such as Borrelia burgdorferi, the causative agent of Lyme disease, Bartonella, a flea-borne bacteria, and Babesia, a tick-borne protozoa similar to the causative agent of Malaria. And one of the more disgusting parasites that uses these mice as hosts is the bot fly.

You may have heard of bot flies in association with people coming back from the tropics with weird bumps, to then have a squishy larva pop out of them a few weeks later (see NBC story here). I had heard of them infecting primates and other tropical vertebrates, but didn’t know they were around in temperate areas as well. But, in our 2009 field season we were finding mice with what looked like tumors or extra testes (they were usually in their lower torso area). The size of the growth in relation to the mouse’s body size is pretty significant, like half the size of its torso in some cases (see below). We really had no idea what these were, so we took some pictures and sent them to John Whitaker, a mammal expert at Indiana State University. He identified the growths as bot flies right away. This year I saw a few more mice with similar infections, and one of the other researchers in my lab did too. And being nerdy scientists, we did some online searching to find out more about this gross parasite that is infecting our animals.

One of the mice we captured in 2009 with a botfly infection. Notice how big it is compared to the size of its body!

The most useful source we found was a Catts 1982 paper in the Annual Review of Entomology. It starts out with a great line:

“Maggots of cuterebrid bot flies are conspicuous, repulsive, and frequently encountered cutaneous parasites of mammals in the New World.”

I don’t know if I’ve seen the word “repulsive” in an academic paper before.* This went through a detailed overview of the life history of this parasite and what is known about different aspects of this larval and adult biology in the Americas. A drawing of a typical bot fly life-cycle is below, and is simply the adult fly lays eggs (1000-3000 per female, depending on species) usually close to host burrows, those eggs hatch into microscopic larvae when they sense and increase in temperature (a potential host), the larva get in to the host through eyes, nose, mouth or open cuts on the skin, they then migrate through the host’s body over a few days and then settle in the abdomen of the host to develop into the final larval stage. They create a “warble pore” through the host’s skin to breathe out of while developing. The larva then emerged out of the pore, develops into a pupae under leaf litter and can overwinter in this stage, and then emerges as an adult. Adults do not eat at all, they only mate and lay eggs.

Illustration of bot fly lifecycle from Catts 1982. The host is on the right side, and two alternate pathways are shown for infection (direct contact with larvae or females laying eggs on host, which is less common).

Yes, this is pretty gross and you may have visions of “Aliens” in your head right now. But surprisingly, it seems like hosts don’t suffer too many long-term effects of the infection. The Catts paper summarizes studies that found hosts have lower than usual protein levels during infection but seem to make up for the loss by eating more. The warble pore usually heals completely in a couple days with little infection, possibly due to compounds in the mucus the larva surrounds itself in.

Last week I did my last field sampling of the year and found a mouse with a really huge bot fly infection. I took a picture to show Angie, the other researcher who had found these infections while doing some mammal trapping. I said it would be really interesting if I caught the same guy again to see if the larva emerged, since it was so big already. 

Animal 595 with a big bot fly infection. The dark circle is the warble pore made by the developing larva. Hopefully you can see how huge this infection is at the time.

And, low and behold, I caught that same mouse the next day and the larvae had emerged. 

Same mouse, no bot fly. The pore is bloody from the larva emerging and his torso looks much more normal-shaped. But the mouse doesn't look too happy!

The little guy did not look like he was in good shape, go figure since a parasite the size of his torso just squirted out of him. When I released him after doing all the handling of the other animals, he just kind of sat on the ground, instead of running away quickly like they usually do. He was all hunched up and kind of wobbled as he walked away. I was even able to reach down and grab him off the ground, which I would never be able to do with a healthy animal. I am thinking he must have been in some significant pain after that lava emerged. I am hoping he is ok, after reading that the wounds heal pretty quickly. Poor little guy! But I guess since I do study parasites, I should be glad that I am studying a host that is so desirable for all these different things, and emphasizes that the host really is a community and that ecology matters at so many different levels.

* If you want to take a look at the paper, there is a lot of other somewhat non-academic language used. It’s kind of funny to see what you could get away with in older publications.

Its just Evolution, not a big deal…

I have been a bit lax on the blog posts lately due to some fall travel and trying to get lab work done before teaching gets too crazy. Speaking of teaching, that is what inspired the subject of today’s post. I will be heavier on the education (less on the musing) today because the biology majors students in our Evolution class seem to have more misconceptions on the basics of evolution than we expected. This prompted me to try to do my part to try and teach some of the non-sciencey people who read this blog a bit about evolution, maybe enough to pass on to someone else or decipher some of the spinning that happens in the popular media.

First, some basic definitions. Evolution is a change in gene frequencies in a population over time. This does not need to occur due to natural selection, it can happen by many different mechanisms. Natural selection as a mechanism for evolution describes that if individuals vary in heritable traits, and if those traits are associated with different probabilities of success in survival and reproduction (i.e. fitness), there will be a shift in the gene frequencies in the next generation to have more of the genes associated with higher fitness.

Natural selection happens to individuals and Evolution happens to populations.

-          Individuals vary and those with poor fitness are selected against (i.e. if you aren’t good at surviving and reproducing you don’t get your genes represented in the next generation).

-          Evolution describes changes in gene frequencies or proportions, so you need to consider more than one individual in order to measure or calculate this change.

Unlike in Pokémon, one individual does not evolve into something else during its lifetime. However, like in X-men, genetic mutations within a population can cause certain individuals to be different, and if there is an advantage to the mutation (say, if being able to control someone’s mind increased your fitness, something Prof. X could have taken advantage of) that mutation would increase in frequency and cause an evolutionary change.

Some things evolution is not:

…directed

·         The process of evolution does not predict the future, i.e. it cannot predict that next year will be particularly dry and have individual plants produce more drought-tolerant offspring. Certain organisms may have more variable genes than others or could be more susceptible to mutation, but these traits may simply allow for rapid adaptation to changing environmental conditions.

…for the good of the species                                

·         Evolutionary changes in a population or species are not always positive or going towards an optimal state that makes a species perfectly suited for its environment. Natural selection does act on individuals that are less-suitable or fit in the current environment, selecting against them, resulting in individuals that are more capable of surviving and reproducing in an environment producing more/better offspring. But, this does not mean there is some guiding force making these changes. There are also negative or neutral processes (genetic drift, bottlenecks, inbreeding depression) that also result in evolution that may not have any positive effects on a population or species. 

It is sometimes hard to think that all the vast diversity of life that we have on earth today, and that has existed in the past, came about by the simple process of some individuals making more or better babies than others in response to the conditions they find themselves in. But there has been a long time for this to happen (we think life emerged somewhere around 3.8 billion years ago) and life or death consequences for what traits an individual has. The power of artificial selection (selection of desired traits by humans) can be seen in creating modern corn from the grass teocinte, broccoli, cauliflower, kale, and almost every other vegetable you’ll see at the grocery store from one primitive cultivar (wild cabbage), and dogs of every shape and size (still all the same species), simply from breeding individuals with desired traits over many generations, exemplifies the amount of genetic variation that can be present in one species or population. Now, think of this same process but the species being selected on are confronted with almost infinite combinations of environmental conditions, competing species, available resources, and random mutations. I think it becomes less crazy to think of evolution leading to all of life on earth once you put it in this kind of perspective.

Modern corn/maize on the left, the primitive cultivar teocinte on the right. A bit different, right?

The thing that is maybe the most important is that variation is a constant in biological systems. Unlike physics, where there are laws that if they are wrong we wouldn’t have things like planets, matter, and the universe, biological processes are fundamentally based in the fact that variation at the individual, population, community and ecosystem level is always present or possible. For me, this makes me think that there is always something totally new around the corner as well sets up a framework for making all kinds of predictions about biological processes.

I hope this has given a good primer to the basic ideas of evolution. I have a couple more things that I think are interesting on the basic subject of how evolution works and why it is important, but I will touch on that in the next post.

Déjà vu and the Balancing Act

Thanks to my brother Jake for altering this comic for me.

We have come to the time of year all grad students dread: the end of the summer, which means the return of the undergrads and our various semester-related duties (classes and/or teaching). In these last few uninterrupted days I am cramming in an immune assay that will be much easier to do when I’m not having to schedule it around meetings and teaching obligations.

This transition along with a New York Times special section on graduate school (came out a few weeks ago, but a professor just brought it to my attention) has me thinking about what the life of a grad student is “normally” like (and I guess when I say normal, I mean during the school year). We are always transitioning between one thing and another, going from teaching a discussion section in the morning to working on a grant application to doing some lab work, having to prioritize what we actually will get done (i.e. what can we half-ass the best today and what is going to take the front burner), with our constant companion being a guilty feeling like we should be working more, especially when we aren’t working and doing something like eating, watching TV, sleeping, or spending time with our spouse.

With any full-time job comes the multi-tasking and extra-busy periods, but I think the biggest difference is with a lot of jobs when you come home you leave your work at work. There isn’t the nagging feeling that you could or should be working on something all the time. Some grad students, they do work all the time, and a lot of them are really happy doing that and often have very productive PhDs as the result. But for those of us that can’t work all the time, it takes a while to find a balance between work and non-work where we don’t feel guilty and still get everything done.

The need for breaks was really solidified for me during pre-lims (part of our qualifying exams). This is a very intense period during our second year in grad school where we have six months to answer four essay questions and then orally defend those questions. There is so much to read and write that you can work all the time, and I did pretty much work on pre-lims whenever I wasn’t in class or sleeping. I did decide that I would take Saturdays off, or at least not work very much those days. I also started doing yoga that semester, which has become a regular part of my routine now. Making the decision to have a break helped be not worry all weekend that I should be working on my pre-lim answers. But, because I was taking a pretty hard class at the same time, I did end up working on the homework for that class with other people on Saturdays, which killed my one free day. The homeworks being really difficult for me would sometimes send me over the edge of stress, making me incapable of working at all that day and undoing any productivity. I felt like a huge bitch on the weeks where I ended up not having a break, even though my friends deny that I actually was.

Now that I am back at the point in the year when I can’t do whatever I need to do whenever I want because of teaching responsibilities and meetings, I need to get back to honing my multi-tasking skills. There is a lot of work to get done and never enough time to do it all. But making down-time one of the things that balances out the week will hopefully make this an efficient and enjoyable semester.

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