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Confocal images of follicle cell stress fibers in stage 13 egg chambers, filamentous actin stained with phalloidin: leftmost depleted of Diaphanous, middle depleted of DAAM, and right w1118 control. Stress fibers in the left and middle images may additionally be affected by the presence of the classic mutant Prickly, associated gene unknown; further work is in progress. Images taken by K. Sherrard, 16 December 2020.

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In praise of roller coaster rides

“...the thousand concurring accidents of such an audacious enterprise….”

-Herman Melville, Moby Dick

Despite what teachers of high school science classes solemnly intone, this business of doing science is the least straightforward endeavor that can possibly be imagined. This was brought home to me in a series of unfortunate events that unfolded this week.

At first, it seemed to be that rare triumph where my simple test of a straightforward prediction actually yielded a clear positive result, instead of the more typical back-to-the-drawing board head-scratcher. If this were a story, that the protagonist was a protein named Diaphanous could serve as a hint that the plot would not prove as solid as one might hope. (Like many genes first discovered in fruit flies, Diaphanous evokes the appearance of animals lacking a functional version of that protein).

The backstory: Lately, my research has been on how stress fibers remodel  to accommodate the movements of migrating cells. But as I work on cells in intact tissues, namely the rind of follicular cells that envelops the developing cluster of cells that give rise to a fruit fly egg, I like to consider the natural experiments that unfold in the course of normal development. For example, these follicle cells migrate for a time, going round and round like hamsters running on a wheel, but then they stop and do other things, like flatten out and secrete the eggshell. They still have stress fibers—these are long contractile bundles of a similar composition to muscle, that help attach the cells to the fibrous surface outside them. But these later-stage stress fibers are much stouter and of somewhat different composition.

I had already established that the stress fibers in the migrating cells depend on an unusual partner, amusingly called DAAM, to form. The more typical protein to help build stress fibers is DAAM’s cousin Diaphanous, but I’d done experiments depleting Diaphanous that clearly showed it was not needed in this case. When I depleted DAAM, though, the stress fibers got really wispy. Oddly enough, I’d noticed that in the much later stages, after the cells stopped migrating, had stress fibers unaffected by loss of DAAM.

So the experiment I wanted to do next was to deplete Diaphanous in the later stages. This was not completely straightforward to execute, though, because I had to avoid depleting it too early. I’d already seen that this caused cells to have trouble with their normal round of cell divisions. It’s a common problem in this sort of work that it can be harder to study later processes if you mess things up before they have begun to happen. The solution makes use of the dazzling array of tissue-specific drivers of gene expression that have been invented for fruit flies. They allow you to drive expression of a gene at specific times and places, targeting particular processes you want to study. To keep a gene from being expressed, you can use something called RNAi, which basically makes a cell chop up the instructions for making a protein sent from the DNA so that protein does not get produced.

In short, I needed a driver that acted late in the follicle cells but not early. Our lab did not have such a driver, since we study the earlier stages. But we’d read a paper with some very clever experiments that made use of just such a late driver, one called Cy2. We requested the fly stock from one of the paper’s authors and she promptly mailed it off to us. Fly researchers are awesomely generous. It’s a tradition that goes back to the earliest days of the field over a century ago to share reagents this way.

Chapter the First: Quarantine. The flies arrived and had to be put in quarantine, out of an abundance of caution concerning the possible introduction of mites into our hundreds of lab stocks. In practice, this consists of isolating the vials on the top of the lab refrigerator. All stocks that arrive from elsewhere must be taken through quarantine, save those from the renowned and very reliably mite-free Bloomington stock center. It meant a delay to the start of my planned experiment, until I could obtain 3rd instar larvae and wash them, a rather amusing exercise on which I have previously posted.

So there the flies sat, two healthy vials with clearly written labels: Cy2/(Cyo); Dr/TM6b. This cryptic shorthand conveyed that along with the driver I’d asked for, the flies conveniently included markers on another chromosome, in case I wanted to build more things into the stock. Annoyingly, they were all senescent adults and developing pupal cases—ideal for surviving the mailing process, but the worst possible stage of colony development for obtaining sufficient larvae for my purposes. I would have to wait several weeks for the new generation to produce larvae I could wash.

In pre-covid times, I could have done the cross right away with existing males, dissecting the offspring on a quarantine-use microscope belonging to a neighboring lab. Normally we share a lot of equipment freely in our department. But the physical distancing requirements have temporarily stopped that sort of thing. And we can’t risk getting mites onto the equipment we use for all our normal work.

To shorten the waiting time (a frequent concern of fruit fly researchers, especially I would think those of us who work on adult rather than embryonic or larval structures, meaning our crosses must extend to the full 10+ days of development time beyond any stock-building that precedes it), I planned to wash enough larvae to siphon off a number of males for the experimental cross. To that end, I also began “blowing up” the stocks I would obtain the females from; I could virgin them ahead of time and have them all ready to go as soon as their husbands emerged from their pupal cases.

When you’re waiting to wash a quarantine stock, impatient for the experiment to begin, they seem to take longer to develop, much like a watched pot. The stock contained the mutation Tubby, which makes for shorter flies but a longer developmental time, so that was part of it. Also room temperature (on top of the fridge) slows development compared to the flies’ optimal temperature of 25 C (that’s 77 to your Fahrenheiters...and to be honest, most of us American scientists are very compartmentalized in their understanding of Celsius; outside of the lab context we speak it no better than the average U.S. citizen). So far, then, the slowness makes sense both physical and psychological. But why the quarantined flies should always produce their burst of 3rd instar larvae on a weekend day, and on the one weekend day I don’t pop into the lab, is more puzzling. But it is the rule, I have found.

I wasn’t going to let it happen this time. I watched them like a hawk (a mosquito hawk?) and sure enough, it was a Sunday when all the larvae began to wander. Wandering larvae is the other, more romantic name for the 3rd instar of Drosophila melanogaster, because they have at last eaten their fill of the mushy rotten fruit they have been burrowing through, and there is nothing else for them to do but come out into the light and air and begin to claim their inheritance as winged creatures of the sky. First, though, they must choose a spot in which to prepare their new bodies. Here in that lab, they climb around on the clean walls of the vial, above the caramel-colored dollop of food, fat, juicy larvae as big as a good-sized grain of rice, big enough to grasp gently in forceps and take through the three ritual baths, soapy water, ethanol, and salty water, that remove any lurking mites or mite eggs from their surfaces. After being placed in a fresh vial and wicked dry with a twist of Kimwipe (lab Kleenex), they will crawl around a bit more, mingling with their certified-mite-free compatriots. In a few more hours they will settle down, stop moving, and let their skins harden into bark. Inside that bark, they pretty much dissolve themselves, save for a few set-aside clusters of cells. They go on to rebuild their bodies into the adult form, complete with intricate jointed legs and multitudinously-faceted eyes and iridescent, cellophane-like wings over the course of about a week (at room temperature).

I spent several hours washing more larvae than usual to establish a clean stock, wanting to have plenty of extra males to father the experimental crosses. If I’d had access to the quarantine microscope, I could have selected extra male larvae—you can already distinguish males and females at this stage-- but it would not really have saved time. I played the numbers game instead. It was a Sunday afternoon, quietest time of the week in lab, and very peaceful. I took my time and changed the bath solutions often to make sure there wasn’t too much soapy water in the ethanol or too much ethanol in the final rinse. I wanted this all to go smoothly with no delays.

I put the now-lawful vial in the 25C incubator to develop, after carefully copying the genotype from the original handwritten labels: Cy2/(Cyo); Dr/TM6b. Incidentally, there are lots of markers of chromosomes, many going back to the original mutations described by early fly workers such as Calvin Bridges and Alfred Sturtevant. They let you follow with visible traits the invisible genes that you wish to follow through the generations. Various labs have their favorite markers, but some such as Cyo (which makes for curly wings) are ubiquitous, and Dr and TM6b were familiar to me as well. Dr (short for Dropped, I don’t know why) makes the eyes very slitted, and TM6b is a whole set of markers that comprises what is called a balancer chromosome: a chromosome that has been scrambled and rearranged so that even though it still has all its genes, they are in the wrong places. This means that none of the usual recombination between sister chromosomes that occurs when egg and sperm form can happen. The advantage to the researcher is that this keeps genes segregated in predictable places. Otherwise, all those markers would not be reliable indicators letting you keep track of the genes you put in place from one generation to another. TM6b can actually include various different markers, but one of them is Tb, easy to recognize in both the shorter larvae and pupal cases and to some extent discernible in adults as well.

Chapter the Second: Cross Purposes. Fast forward two weeks (you can—I sadly could not—this being November of 2020, I would certainly have appreciated the distraction). So I waited, none too patiently, for the new adults to emerge. Meanwhile, I tended the stocks I would virgin for females: two different RNAi lines for Diaphanous and one, a control, for its cousin DAAM which I already knew was not required for the later-stage stress fibers. I built up a collection of ladies in waiting, captured shortly after their eclosion and isolated in vials away from all male contact, so I could be sure their offspring would be the genotype I wanted. [A note about the term ‘eclosion’: one might be tempted to call the emergence of the adults from their pupal cases ‘hatching’, but that term is reserved for the larvae coming out their eggshell. You only hatch once, even in the doubled lifestyle of these metamorphosing beasties.]

Finally the washed flies began to eclose. All my usable Cy2 flies were in that one vial. I briefly knocked them out with carbon dioxide gas, used a fine paintbrush to separate the males, and added 3 males each to the three bevvies of expectant females. There were still a few males left, enough to establish the new stock of Cy2 for future use.

At last, more than a month after conceiving it, I’d begun the experimental cross. It would be two more weeks before I had the flies to dissect and the beginnings of an answer. Fly work involves a lot of waiting, and to cope with that we tend to have a lot of irons in the fire. All that juggling can be rather distracting. Sometimes, depending on how other experiments have gone in the interim, I’ve unfortunately moved on from the original urgency of a question by the time the flies are ready to examine. It’s a hazard of the work.

Though I did not yet realize it, I’d made two mistakes. First, I should have looked a bit more carefully at those Cy flies. Second, I should have done the proper control. Sure, crossing them to the DAAM flies was a pretty good control, but there was an even stricter one, that tested whether the driver stock alone had any effect (it should not, but you like to be sure). I should have crossed the Cy2 flies to what we call wild-type, a stock called w1118 that has white eyes, incidentally [link] the first fly mutant ever identified and the foundation of fly genetics.

I hadn’t wanted to use up any more of my precious males, and figured I could always do that control later, if the experiment turned out promising. A lot of us cut corners that way, and it isn’t necessarily less efficient. But sometimes it snarls you up and wastes your time instead of saving it, and makes you go through all sorts of contortions trying to make sense of your data with less information than you should have had.

Chapter the Third: The Experiment. I waited out that two weeks, pursuing other work and trying not to pay too much attention to the news. I wore my mask and stayed in touch with my loved ones over zoom and the like. I hung up bird feeders to entertain my cats and my family alike. I went on long walks by the lake. Time passed. At last the grand day arrived: my experimental flies had begun to eclose. I gassed them and tapped them out of the CO2 pad. Now here was a wrinkle I’d shoved to the back of my mind: those extra markers that I didn’t need, the Dr and TM6b. In a clean experiment I’d have gotten rid of them, but that would have required another couple generations. I’d wanted a quick provisional answer, in order to decide whether it was worth the time and trouble to do the more careful version of the experiment. So: would I dissect the TM6b-carrying flies, or the Dr-carrying flies? It had to be one or the other. The balancer chromosome carries a number of mutations so it would be more likely to do something weird to the cells I was interested in. Not that that was very likely, but I might as well be careful. Dr it was then: that only affected the eyes, as far as I knew. What were the chances it would mess up my experiment on stress fibers in follicle cells?

But none of the flies had Dr eyes. That was odd. I looked closer. Half of them sure looked like Tb flies, shorter and a bit chubbier, though you never want to depend on your ability to discern that marker in adults. The others, the longer ones? They did have some oddly short hairs on their dorsal thorax (around the back of the lower neck, if you want to be anthropomorphic about it), much shorter than the clipped ones you see with the marker Stubble. It kind of reminded me of a marker I’d seen once or twice. Well, that must be what these were; maybe the label had been written wrong.

Impatient to get the experiment done, I swept the short-haired flies into a fresh vial with a bit of yeast. The yeast was to encourage egg production (they’re called fruit flies or vinegar flies, but it’s really the yeast on the rotting fruit that they’re after). I added a few males which were there for the same end. You could say the way to a fine set of ovaries is through both the heart and the stomach. Two more days to go before the dissection. For good measure I put some plain-vanilla w1118 flies on yeast to serve as extra controls.

On the appointed day, I got out my fiercely pointed #55 forceps and began the dissection. I nearly messed up by dissecting the early stages by habit—the technique to do so destroys most of the older egg chambers—but luckily remembered what I was about it time, and switched to the method to optimize acquisition of undamaged later stages. I fixed for 15 minutes in 4% paraformaldehyde, rinsed three times in phosphate-buffered saline solution with Triton-X detergent, and added a stain that would label the filamentous actin, the principle component of stress fibers among many other cellular structures. I put it in the lab fridge (the one where no food is allowed!) to stain overnight. The next morning, early, I came in and rinsed off the stain and made slides. Then I went to the womb-like room where one of my favorite workhouse microscope lives, the renowned Nikon 800 laser scanning confocal microscope. I did the necessary 2020 ritual wipe-down of all surfaces with 70% ethanol, and fired her up.

And oh, it was beautiful. I was so disciplined; I began with the controls to set up the correct laser intensity and gain at which to collect all the images, so the brighter ones would not be out of the range of measurable brightness and everything could be properly quantified. But it was already clear from the what I saw on the computer screen as I centered examples, focused, and took images that the experimental egg chambers had strongly reduced stress fibers. I took lots of pictures, happy that for once my experiment had gone as planned and given me a clear answer.

Also, can I just say how much I love the stain Oregon Green phalloidin? The name itself is lovely: as a native of the Pacific northwest I find it so evocative: the green of deep cushiony moss and ferns and forests of hemlock and douglas firs; and phalloidin itself is a stain derived from mushrooms with which those forests are rife. (Phalloidin, now there’s a scary toxin: it binds so tightly to filamentous actin that it stops your heart. Unlike a lot of other toxins, it doesn’t make you nauseated, so you absorb it until it’s too late for any antidote. But that’s why it’s such a good stain. You just have to wear gloves, or wash your hands after pipetting it. And we all wash our hands so often nowadays it makes no never mind.) There’s red phalloidin, and far-red phalloidin, and even ultraviolet phalloidin (but most microscopes don’t have the right filter sets to light it up very well): but green phalloidin is the king as far as I’m concerned. So bright, and a short enough wavelength (only 488 nanometers, vs. 566 or 647) that it shows up structures the more finely. You can definitely see the difference: it’s sharp as can be.

So, I had the preliminary results I had hoped for: the Diaphanous flies had reduced stress fibers. It doesn’t actually happen to me all that often, that I get a clear answer, either what I predicted or the opposite which is almost as good in science. At least that’s progress, an increase in understanding. No, usually I stumble over these head-scratchers of outcomes. Interesting results, but interesting in a complicated way that require a lot more work to make sense of, if you ever do. It’s partly down to most of my experiments involving imaging with a microscope: you get a lot of unexpected information that way, if you keep your eyes open. But it’s also that I seem to be attracted to the sort of problem that does not yield neat answers—the way some people are attracted to overly hairy guys on motorcycles who are a bit too into mild-altering substances and petty crime. I think I’m the one to straighten them out, but usually I’m the one who gets burned. But this time I had prevailed!

This was just a start; of course I needed to replicate, do some more dissections, get more numbers, reach levels of statistical unassailibility. In particular, I didn’t have as many clear examples of the DAAM control as I needed. Also, I’d do the proper control, and maybe even un-double-balance that Cy2 stock to get rid of the pesky extra markers.

Chapter the Fourth: The morning after. Yeah, and now I’d better take the time to figure out what is going on with that marker that is not Dr. Because, unlikely as it was, wouldn’t it be a shame if it were somehow affecting my results? Worst-case scenario—because that’s how we self-questioning scientists have to operate, ever since the dawn of time or at least the Enlightenment—worst-case scenario, then, is this marker, whatever it is, is the thing responsible for the reduction in stress fibers. Oh, but that’s very unlikely, I tell myself. Besides, the DAAM controls didn’t have reduced stress fibers.

I looked at the original handwritten label, still on the vial of flies on top of the fridge in quarantine. Maybe that D might actually be a P. What was Pr? I’d never heard of it.

I went to the master compendium of fruit fly genetics, FlyBase.org, and looked up Pr. Purple, an eye color gene on the first chromosome. I was looking for a gene on the third chromosome, so that couldn’t be it. I tried a different approach: I DuckDuckWent (DuckDuckGoed doesn’t sound right; if you haven’t heard of it, it’s a more private alternative to Google) images of Drosophila markers. There was that classic poster I’ve seen hanging in various labs, of the most common markers. And there was that marker I’d been reminded of, with the very short hairs. Sn it was called. Could that be my marker? It would have to be some pretty bad handwriting, to make an S look like a D; r to n is easier to imagine.

I went back to FlyBase and looked up Sn. It was the gene Singed. Like if you got to close to the outdoor fire pit on the patio (a way to safely hang out with your friends outdoors even during the Chicago winter), and singed your eyebrows most of the way off (and no, I haven’t done that yet). Also on the first chromosome, though. But look here, this is interesting: Singed is an actin-bundling protein. I read further down the page that summarized the work of dozens or hundreds of researchers over the decades. Yes, it was expressed in the ovaries, and yes, it was known to affect stress fibers. That would be worrying if it were my marker. Lucky it’s not.

I wasn’t getting anywhere. I tried yet another method, going to the webpage for the Bloomington stock center. It’s very well organized, and they have a page showing the details of all the balancer stocks they keep. There ought to be a clue here, for any marker that a researcher could assume another lab would recognize. I go down the list to the TM6b stocks, and find it. Pri, aka Pr, for Prickly. Causes short thoracic bristles. That’s my guy.

Back on FlyBase, I learn that Prickly is one of the classic mutants discovered in the early days of fly research. And this is weird: it has not been annotated. That is, nobody has figured out what gene it is a mutation of, let alone what biological processes it participates in or what tissues it’s expressed in (this matters because if it’s not active in the follicle cells, my experiment would still be valid). They could; it’s a straightforward enough task given that the whole genome is sequenced, but apparently it’s not one that anyone’s found worthwhile. So all we know is it makes very short, deformed bristles that look to me a lot like those of Sn.

Okay, now I am getting worried. What are the chances that this is NOT a protein that affects something like actin bundling and therefore messes up stress fibers? Maybe I had only seen what I wanted to see with the DAAM control. That’s a hazard of doing science, because it’s a hazard of being human. That’s why controls are so important. I consider my experiment in this new and harsher light. Maybe the Diaphanous results are just a phantom of wish fulfillment, summoned by this Prickly hitchhiker I’d never meant to take along for the ride.

I’d already begun the proper control that would answer this question, but meanwhile, while I wait for those flies to emerge, is there anything else I can do? Maybe I should dissect those formerly scorned Tubby flies; at least they lack Prickly. But according to the list at Bloomington, that particular stock has a number of other mutations on its TM6b chromosome, including one called Bri. Bri is a twin of Pri in more ways than one: it also causes very short bristles, and is also unannotated so we have no idea what protein it makes or when or where it acts in the body. Without asking the researchers who sent me the flies, I had no way of knowing if Bri was in there or not.

It would be a bit awkward quizzing them about their flies. We all tend to overdo the shorthand in labeling our stocks, and don’t always remember all the extra mutations lurking there. It’s tripped me up before, when I uncovered interacting mutations I hadn’t known to worry about until they unhinged my crosses. Don’t get me started on vermillian eye color: it’s a real bear. Either way, I’d have to check the controls and unbalance the stock to have a real answer, so probably better not to pester them.

I can’t resist having a quick peek at the TM6b flies though; I’ll be dissecting them tomorrow and should know by Sunday or Monday if the Diaphanous results are evaporating or not...that is, if Bri or something else is not further muddying the waters. A positive result would be definitive; a negative one will require further research. Well, either one will require further research, but one will be more cheerful and the other more like putting nails in a coffin of my hopes one more time. And that, my friends, is what it’s like to do science. (At least I get to see more Oregon green on the confocal, though).

Epilogue. What lessons can we draw from this (mis)adventure, this stomach-churning roller coaster ride of thrills and doubts that is my life in science?

1. Do the proper controls from the beginning. (Although that would have cut out the thrills as well as the doubts, so to be honest, I’m not totally on board with this one).

2. Take the time to look at the flies you are about to cross, and make sure they have the markers you expect. Harder, probably unrealistically hard, is to make sure they don’t have the markers you don’t expect. That would require a Rumsfeldian level of perceiving unknowns unknowns.

3. Remember the limitations of shorthand for conveying a genotype, which like the face we present to the world is invariably far more complex than there is room enough and time to write out.

4. Murphy’s law reigns supreme in this world of ours. What were the chances that the unwanted marker  I’d thought I could ignore for a first-pass experiment would turn out to be a different marker I’d never heard of that might  affect stress fibers in my cells? Still, it made for a good story, which I haven’t come across in all this interminable slog of an Autumn.

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From Zoologische Wandtafeln, by Dr. Rudolf L. Leuckart, Table XLI of Gall-forming Hymenoptera, #8 and #16 (published by Theodor Fischer in Cassel). See also Nature vol. 68, page 319 (1903), https://doi.org/10.1038/068319d0

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Safe Havens

“...those enviable little tents or pulpits, called crow’s-nests, in which the look-outs of a Greenland whaler are protected from the inclement weather of the frozen seas.” -Herman Melville, Moby Dick

‘Gall’ is a word evoking bitterness and pain, as people entishly take the side of the trees thus injured by their tiny animal denizens. Yet to a mite or a mother wasp, for whom a tree is an entire world, a gall is a shelter grown whole-cloth from leaf, stem, or bark by means of a chemical nudge. Within this cozy refuge the creature can feed at leisure on provisions filched from the plant, safe from wind, rain, and all but the most determined of predators.

Galls take a great many forms, from reddish protrusions bugling up from the tops of linden or alder leaves, to smooth-skinned, foam-cored oak apples and pale pink hair-lined knobs crowding the leaves’ surface. Pretty much any odd protuberance you see on a plant that looks unharmonious to the whole is likely to be a gall. Each type has a characteristic instigator, most often an insect or arachnid, which often confines itself to a single type of plant. Often these are larvae undergoing their development in the safest of nurseries. Others may move in and join them; to make oneself at home in a place built by someone else is to be inquiline.

Once I began to take note of galls, I started to see them everywhere on my walks about the city. The world works like that: fractally obliging by opening up new universes to our awareness if we give the slightest tug to the veil. Even though there is always much to be learned at the library, or on the internet, this time I wanted to discover things using only my hands and eyes. How many different kinds could I find, for one thing. Half a dozen the first afternoon – the first right across the street from my house, in a linden tree. I found more in the scrubby semi-wilderness of untended parkland to the east and to the west of my neighborhood. The diverse set of trees on campus are perhaps too well-cared for to yield many galls, though I did find some.

With a borrowed dissecting microscope I examined the galls, admiring their intricate textures echoing details of leaf and stem. I cut open a few of them to see who was inside, but regretted it as antithetical to a contemplation of these small shelters, and soon desisted. Likewise, after a while I stopped plucking my finds to bring them home. Instead, I took to examining them in the field with unaided eye (being at that fulcrum of middle age where my nearsightedness is intersecting with loss of acuity such that looking at objects a few inches from my face provides an amazingly clear view, one that I fear may soon be no longer available to me). No, this is not Biology – rather, it is a way of relishing life in all its resilience and ingenuity.

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The last piece of data I collected before our shelter in place began, imaged on March 4: Laser-scanning confocal micrograph showing the basal surface of follicle cells from a late-stage egg chamber. Cyan is phalloidin-labeled f-Actin on stress fibers and magenta is the cell-matrix adhesion protein Paxillin, tagged with yellow fluorescent protein.

At this stage, the follicle cells which surround what will become the egg are stretched very thin, and have nearly finished secreting the many layers of wax and chitin that form the eggshell. Earlier, they had aligned to march in step around and around the egg-to-be, depositing trails of matrix that formed a corset making the egg adopt its properly elongated form. But by the stage shown here, they are oriented every which way, holding on with all their might.

"Surely all this is not without meaning."

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Lab in the time of covid-19

"...since the epidemic had broken out, he carried a higher hand than ever; declaring that the plague, as he called it, was at his sole command; nor should it be stayed but according to his good pleasure. The sailors, mostly poor devils, cringed, and some of them fawned before him; in obedience to his instructions, sometimes rendering him personal homage, as to a god. Such things may seem incredible; but, however wondrous, they are true. Nor is the history of fanatics half so striking in respect to the measureless self-deception of the fanatic himself, as his measureless power of deceiving and bedevilling so many others."

-Herman Melville

These days I go into lab once or twice a week. The plants I brought home in March in a massive rescue operation are back on the broad, light-filled windowsills. Once again they thrive in the peaceful assurance that they will not  be chewed on by bored cats. Lab is almost as quiet as it used to be when I would go in as early in the morning as I could manage (nowadays, I prefer to reserve early mornings for walks or runs, before the summer heat gets too bad). For me, it has always been a relief to turn to lab work as a break from the hard intellectual labor of planning and analyzing experiments and placing your findings in the context of the published scientific literature; it is hard to escape the impression that there are just too many biologists making far too many discoveries nowadays to have much hope of making sense of anything. If the ratio of data to story gets much higher, I fear we’ll have to cede the project of interpretation to pattern-detecting software, which would take much of the fun out the whole enterprise.

In any case, one of the great pleasures of doing biology is that you get to use your hands for part of the work. Not so much in recent months, however. During the shelter in place and exile from lab that my university experienced from March-June, all that was left was the intellectual work of reading, thinking, analyzing, writing, attending meetings and  talking with colleagues by zoom. It's unbalanced to have all your work take place within the narrow, two-dimensional confines of a computer screen. So I am very grateful to be back to doing experiments with flies.

Many fly experiments start with a cross of two different stocks to get progeny one or more generations down the line with certain desired characteristics, such as a particular protein lighting up with a fluorescent label in a particular tissue at a particular time. So you sit at a microscope and knock out flies with carbon dioxide gas, and gently push them around with a paintbrush to separate males from females and virgin females from experienced ones. How can you tell? You keep the females who look paler and softer than their compatriots, because they emerged from their pupal cases within the past 20 minutes or so, and assume all the others have been around the block. And why do only the females need to be virgins? For the exact same reason that people in many times and places have cared about this.

For more involved crosses, you will be selecting for or against various visible markers to assist sorting out your desired genotypes: stubbly vs. long hairs on the dorsal thorax; stubbly vs. long or abundant vs. sparse hairs on the lateral front thorax (reminding me of an old Seattle joke in poor taste: How do tell the bride at a Swedish wedding? She’s the one with braided armpit hairs); wings straight or so curly the flies should be called crawls because they cannot fly anymore; eyes white or orange or brick red or many shades between, including some only visible by fluorescent light—just to name a few. I’m convinced that looking hard at anything is one form of meditation, and we Drosophologists get to know our flies very well. Then again, there is the early-grade-school satisfaction of circling and crossing out: keep these flies, discard those. This is algebra embodied in living, breeding organisms. In certain types of crosses, we can see direct evidence of that fundamental generator of evolutionary diversity and sexual healing, meiotic crossing over.

In the old, pre-pandemic days, this work took place in a communal “fly room,” where a half-dozen different labs kept stations for fly work. There were two radios, usually both turned on and both playing NPR. I don’t know how it was for others, but my attention to the news stories wandered in and out depending on how complicated my crosses were to sort out. Sometimes someone would turn the volume way down in order to have a conversation, maybe half the time to do with science and the other half something else.

Now, the labs have set up fly stations in their private spaces, where access can be limited to one or two people, minimizing airborne exposure to the virus and reducing the need to exhaustively clean the scopes between users. It’s yet another of the ways this pandemic has isolated us from our habitual social spaces. I hear there’s a Slack for former fly room aficionados, but I don’t have the bandwidth for that these days: I am spending much of my free time keeping up with extended family and a few close friends near and far (since that distinction hardly matters any more, for better and for worse).

Today, working at my lonely fly station in a quiet lab, I put NPR on, streaming it from my laptop into my headphones. We used to be asked to refrain from wearing headphones or earbuds while in lab during regular hours, as it discouraged conversation and normal interaction. Now, of course, none of that can be helped in any case. Despite the often worrying content of the news—covid cases rising in the majority of U.S. states, most horrifically in Florida, governor of Oklahoma tested positive; Greece now requiring tourists provide proof of recent negative test; White House attempting to roll back climate-change mitigating regulations on infrastructure projects; Ruth Bader Ginsburg in the hospital for an unspecified infection (but also: legitimacy and extent of reservations in Oklahoma resoundingly upheld by the Supreme Court, in a rare promise kept to Native Americans; public radio examines its own racial biases in the workplace; White House gives up on demanding colleges and universities hold in-person classes or have their foreign students lose their visas)--despite this, it was a wholly comforting experience, to sit in the air-conditioned lab listening to calm, reasoned voices sort through local, national, and international news while putting my flies in order.

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An ovariole of egg chambers at different stages of development towards the Drosophila egg. Confocal micrograph image by K. Sherrard.

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Part III. Larval washes

I have taken a circuitous route, but I am now ready to describe how one washes a Drosophila larva of any unwanted companions. I recently was given several fly stocks from two labs, one in California and one in Hungary, in the generous collaborative spirit that has characterized fly work from its earliest days. Like many another fine thing, flies are augmented rather than diminished when you share them with someone else; and people who work on them have generally assumed the same holds true for the scientific discoveries they inspire.

As a matter of routine procedure we must quarantine any such new arrivals. When the third instar larvae crawl out from the gooey mess of yeast, molasses, apple juice, and cornmeal that is both room and board, and prepare to stick themselves to the sides of the vial and transform their cuticle into a sarcophagus and their insides into a pulpy mess, to self-resurrect five days later as adult flies, that is the time to wash them in three successive ritual baths, after which they will be permitted to join the rest of the crew on the common berth of shelves.

I take a blunt pair of forceps, and gently pick up a likely-looking grub  who’s wandering about on the inside of the vial wall. I have three small petri dishes laid out on the lab bench, and I dip him into the first one, soapy saltwater (isotonic with body fluids to minimize osmotic stress). It rinses off a layer of debris.

After a moment I transfer him to the middle dish. Now this is a dip fit for a sailor! 140-proof grog, or as we usually call it in the lab, 70% ethanol.  He won’t last long here, so I quickly pluck him out and move  him to the last dish, a gentle salty rinse, this time without the soap. On hitting this bath he briefly zips off like a rocket, then settles down.

I move him into a fresh vial, a dab with a twisted bit of tissue to dry him off, and he’s left in peace to get back to the all-consuming business of metamorphosis. Another dozen or so likely young grubs of both male and female (you can see the difference, though usually random choice works out fine), and in less than a week, I’ll have a new, clean stock for my experiments.

It’s no fish story that the grubs rocket when they hit the third bath. The reason has to do with the aforementioned boundary layers. When I transferred the grub from the ethanol to the salt water, a thin layer of ethanol remained trapped around him. When he touched the surface of the salt water, the weaker surface tension of the ethanol made him shoot away.

Or so I assume from what I know of fluid mechanics. I should stop and to consider what the alternative explanations could be. It’s possible that the zipping is due to some other mechanism than surface tension. However, the observation that putting larvae directly into salt water has no such effect suggests the transfer from ethanol is important. Alternately, it could be that the larva is actively locomoting in the salt water. Though why it would not do so in the other two liquids is puzzling. It’s also a more rapid movement than I think they’re capable of. To disprove the active locomotion hypothesis, I could dip killed larvae (or better yet grains of rice) in ethanol and then in salt water.

Since we’ve arrived at fluid dynamics, which among other lessons teaches us that the world the great whale swims in is fundamentally different from the microscopic world of lice and yet more microscopic creatures, I’ll end with a fluid-mechanical variation on Jonathan Swift’s verse.

     Big whirls have little whirls      That feed on their velocity,      And little whirls have lesser whirls      And so on to viscosity.          -Lewis Fry Richardson, mathematician and meteorologist

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Part II. Larval washes

Whales might have escaped lice by their return to the primeval salty seas (for very few insects can survive in salt water). There are creatures which go by the appellation marine lice or whale lice, but these are in fact amphipod crustaceans, not insects. Small comfort to the whale who harbors them in its nostrils, eyes, genital folds, or skin lesions! The parasites must find a non-smooth surface to cling to, after all, and whales are on the whole very streamlined. The flattened hitchhikers are correspondingly larger than our rice-grain sized fellow travelers, being up to an inch long.

You have to pity the poor whale, fingers buried in the mitten of their aquatic wing of a hand, unable to scratch at these unwelcome intruders! You might think that rapid swimming could dislodge them, but there is a strange phenomenon of fluid mechanics called the Boundary layer (that same as that which permits dust to collect on whirring fan blades) which protects the lice from such simple maneuvers. We’ll get back to boundary layers momentarily.

As with terrestrial mammals and birds, species of these parasites are unique to species of whale. As it happens, the sperm whale takes it even further, with males suffering from Cyamus catadontis and females and calves from Neocyamus physeteris. This odd specialization might be due to males spending more time in colder polar habitats, whereas females tend to stay in more equitable latitudes. How strikingly this mirrors the situation for the Nantucket whaling men, who plied the waters of the great antarctic sperm whale fishery for years-long voyages, leaving wife and children to farm the sandy soil of Cape Cod and the islands.

As for the fly, lice are far too large to trouble them as parasites, but they have their mites to afflict them. Or, more to the concern of Drosophologists like myself, to afflict the researcher. Mites, incidentally, are neither crustacean nor insect, but part of yet another great and venerable order of arthropods, the chelicerates, which also includes spiders and horseshoe crabs. A variety of mites can infest lab cultures of flies, eating their food or even their eggs. A bad infestation can wipe out precious stocks, costing months or years of work.

How many unique fly stocks currently exist is an interesting question. One major stock center, in Bloomington, Indiana, had 67,634 stocks in 2017, all donated by scientists to share with their colleagues; according to their website, they mailed out 218,429 subcultures in 2017, for a nominal fee. But researchers routinely create or combine stocks of their own, and these must be taken care of if they are not to be lost. In practice, this means “flipping” flies to a new vial of food about once a month. Because the fly life cycle is faster than that of any of the mites that afflict it, this prevents the mites from gaining a toe-hold (or, I should say, a tarsal-hold).

Unlike bacteria or C. elegans, fly stocks cannot be frozen down and stored until needed. This means that virtually every fly stock currently in use was descended in an unbroken line from vials kept in the renowned lab of T. H. Morgan. And where did he get his flies from? No one is sure: he is known to have encouraged his student Fernandus Payne to collect them from the wild, by means of placing ripe bananas on a sunny windowsill. But there is some evidence that he himself, upon embarking on his work with Drosophila, requested a vial of inbred flies from one of his colleagues who’d been working on them. An inbred line would have been more useful in the search for newly acquired mutations. In any case, it is certain that virtually all stocks of flies now being used in the lab are descended from Morgan’s original flies, through some 2000 generations of life in captivity. Oddly enough, this is the evolutionary equivalent of 60,000 human years, or about the time we started wearing clothes, according to the testimony of our lice.

Yet again to be continued. . . .

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Drawing of a louse clinging to a human hair, by the polymath and microscopist Robert Hooke. From MICROGRAPHIA or some physiological descriptions of minute bodies made by magnifying glasses with observations and inquiries thereupon, 1665.

Doesn’t this fellow look like a sailor clapping on bravely to a line high above the deck of a pitching ship, while a mate shouts orders to furl a sail? It can’t be a restful life, being a louse. Though at least they have six limbs to grasp with!

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Larval washes

“So, naturalists observe, a flea Has smaller fleas that on him prey; And these have smaller still to bite 'em, And so proceed ad infinitum. Thus every poet in his kind Is bit by him that comes behind.”       -Jonathon Swift

I wanted to write about a necessary task in the fly lab that I’ve been doing lately, namely washing some newly acquired fly stocks clean of whatever parasites they might be harboring. We run a clean ship in our lab. But much to my surprise, there is no mention in Moby Dick of the lice that habitually infested sailors, so I have drawn my net up empty in my search for an apt quotation.

If you will grant me poetic license (the true scientist, I maintain, partaking not a little of the poet’s nature), I will draw the connection myself, in showing you how we are all plagued by such unwelcome hitchhikers, from the great whale through middling humans and down to the tiny fly. Be forewarned: once given such a license, I am apt to go freewheeling around and following the strangest eddies of thought. For once we begin to consider the louse, and the still smaller (as appropriate to our relative scales) mites that can infest fruit fly cultures, we will find irresistible avenues unfurling before us, beckoning exploration. Some faulted Melville for such antics: I say, let such show themselves out the nearest exit and find a straight and narrow corridor more suited to their goal-oriented love of efficiency. Curb your enthusiasm? Why not crawl into your coffin ahead of time and seal it up, lest you be distracted by the March clouds winging their way across the changeable skies?

Lice are insects of the order Pthiraptera, whose closest familiar relatives are the true bugs and the cockroaches. They are obligate parasites (that is, with no other way to make a living) on warm-blooded animals.  Virtually every species of bird and mammal hosts their own particular species of lice – including even Antarctic penguins (but for some reason excluding bats, monotremes such as the platypus, and the scaly pangolin).

Humans harbor three distinct species of lice: head lice, body lice, and pubic lice. Given the specificity of host-parasite relationship, there is a wealth of evolutionary information to be gleaned from these unsavory characters. Unsurprisingly, the head and body lice are most closely related to those of our closest living relative, the chimpanzee, with a divergence time matching our divergence from that species about 6 million years ago. For reasons it may be better not to speculate too deeply on, however, the pubic lice, Pthirus pubis, were apparently acquired from the gorilla only 3 million years ago.

It is our species of body louse, Pediculus humanus corporis, that has provided some of the most fascinating information about a largely unknowable period of human history, before the cave paintings of 30,0000 years ago, but after we became Homo sapiens, some 200,000 years ago. When did people begin wearing clothing? If you don’t get your scientific answers from Genesis, it would seem a hopeless question. No cloth or hide covering could last remotely that long.

Human body lice evolved from human head lice. They are specialized to live in our clothing, and are unable survive on naked humans. Thus an estimate of the time they split off evolutionarily from our head lice provides a neat piece of evidence as to when humans began wearing clothing. This turns out to be somewhere between 80,000 - 170,000 years ago, well before we left Africa. See Reed et al. (2007) for more information.

To be continued….

References

Reed D.L.; Light, J.E.; Allen, J.M.; Kirchman, J.J. (2007). "Pair of lice lost or parasites regained: the evolutionary history of anthropoid primate lice". BMC Biology. 5 (7)

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Camera-lucida drawing of an etherized living fly by Edith M. Wallace; from Contributions to the Genetics of Drosophila melanogaster (Calvin Bridges, Thomas Hunt Morgan, and A. H. Sturtevant, 1919).

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Eye of the Fly

“What the white whale was to Ahab, has been hinted; what, at times, he was to me, yet remains unsaid.” -Herman Melville

When you look into the eye of a fly through the world-changing view of a microscope lens, you see a multifaceted oval almost as large as the head itself, wreathed by a few scraggly hairs. Their color, especially in the younger flies, is enchanting and vivid, ranging from palest apricot through vibrant orange to a rich ruby or still darker vermellion. So-called wild-type flies such as Canton S or Oregon Red have eyes of a more natural-looking brick red. However, the strain of flies most often used as the so-called wild-type is one whose eyes are white.

The mutation causing white eyes was the second one ever discovered in Drosophila, found in 1910 by Thomas Hunt Morgan. Mendel’s disregarded work had recently been rediscovered, and the quest for the physical basis for inheritance was on. Morgan was skeptical of Darwin’s theory of Evolution by Natural Selection, as were many biologists in the early 20th century. The sort of continuous variation that Darwin emphasized had not yet been shown to be heritable.

For two years Morgan’s lab had been searching without success for heritable mutations. This was decades before x-rays or chemicals began to be used to induce mutations for experiments, so they had to wait for a low-frequency natural and non-lethal mutation to appear. Although fruit flies have a marvelously complex external form that allows plenty of scope for recognizing morphological mutations, they are very small. What’s more, they have a most maddening tendency to fly away when the cotton stopper is pulled from the vial in which they make their laboratory home. In short, they must be anaesthetized and examined under a microscope. In the old days researchers used ether, though the fumes were harmful to both fly and researcher. Nowadays we mostly use carbon dioxide, which is harmless (except when added to the atmosphere in large amounts; fortunately, our species is far too wise to do something so self-destructive).

Two years. It must have seemed a long time to be looking at nothing but ordinary-looking, run-of-the-mill, wild-type flies. Oh, there must have been mutants they missed; ones they’d have spotted if had they come along a bit later, after they’d gotten good at recognizing the little critters. They must have seen flies in their dreams every night; the sheen of wings, the exact patterns of hairs around the eyes and on the thorax. The more they looked, the more they would have seen: that’s how it always is.

So they persisted, and at long last they found a white-eyed fly. white^1 is the mutant allele that got genetics barreling full steam ahead. It was recessive: like Mendel’s white pea-flowers, or human blue eyes, it only showed up if both parents had the trait. But there was a twist. Half the males whose mother had one copy of the white^1 allele and one copy of the wild-type red one came out with white eyes, regardless of their father’s eye color. Morgan had not only found a heritable mutation, he had found a sex-linked one.

To this day white-eyed flies are of fundamental use in Drosophila research. They are the control in many experiments, as well as the starting point for a novel mutation or genetic insertion (whether by CRISPR or p-element insertion). Successful insertion into the genome is detected by the appearance of red eye color in the offspring. As delighted as T. H. Morgan must have been to spot that first white-eyed fly, nowadays researchers are more apt to be thrilled by the appearance of a red-eyed fly among a sea of white.

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Camera-lucida drawing of an etherized living fly by Edith M. Wallace; from Contributions to the Genetics of Drosophila melanogaster (Calvin Bridges, Thomas Hunt Morgan, and A. H. Sturtevant, 1919).

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Virginning

“. . .hinting that his ship was indeed what in the Fishery is technically called a clean one (that is, an empty one), well deserving the name of Jungfrau or the Virgin.” -Herman Melville, Moby Dick

One of the first skills any new worker on fruit flies learns is how to “virgin” flies. The term, though  at first startling, quickly becomes a routine bit of background vocabulary.

To virgin flies you have to be able to make two distinctions: that between male and female, and that between youth and age. The virgin part, of course, refers only to the females. I’m sorry to have to tell you that the tenets of sexism are deeply entrenched in biology – which is not to say that we must accept them in the world of people. On the contrary, to a large extent the long history of humanity has been unchaining ourselves from the demands of mere biology.

For breeding crosses, however, we can accept only the most tender and nubile females, only just emerged from their sarcophagal pupal cases and unfurling their opalescent wings for the first time. On the other hand, any decrepit old males will do for the fathers, be they as shrunken-bellied and disfigured as Ahab himself, limping beside his glowing young bride.

Why the strictures on the females? In controlled breeding crosses that are the heart of most fly work, where we wish to combine several sets of traits in the experimental offspring, we must know unequivocally who the fathers are. And the only practical way to do that is to isolate the mothers-to-be before they have a chance to accept sperm from whatever random fly whirs his wings at her in the privacy of their home vial.

It is easy to tell female from male fruit flies under a dissecting microscope. The males have black hairy butts, and females do not. Females are also larger, befitting their need to produce a great many eggs. Students get taught other subtleties, such as to call the butt hairs “external genetalia,” and about the sex combs on the male’s forelimbs and so on. But the gestalt quickly becomes quite obvious.

Youth, too, is easy to recognize in a fly. Even after their wings have unfurled, the newly eclosed flies are paler, with a dark spot on the left side of their underbelly, and a soft, wrinkly appearance from their cuticle having yet to harden. When the spot disappears and the wrinkles firm up, you can still distinguish that day’s newly emerged from those that were born yesterday, but you risk accepting one  no longer virgo intacto, to the possible detriment of your experiment.

The most common mistake a rookie makes calls to mind an ugly feature of the old days of sailing ships, the unsavory fate of the pretty young cabin boy. A newly emerged male for a few brief moments looks very much like a fair young princess. Yet the fate of such flies in the hands of a careless or inexperienced fly worker is nothing to bemoan. Rather it is a brief worldly paradise: a harem of virgin females all to himself, the father of nations. . . at least until the hapless researcher discovers the mistake and tosses the vial into the trash, where the little world will eventually succumb to ecological collapse.

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Why flies?

“To write a mighty book, you must choose a mighty theme. No great and enduring volume can ever be written on the flea, though many there be that have tried it.” -Herman Melville, Moby Dick

Why flies? Whales are an ideal symbol for the grand explorations of the 19th century, whereas fruit flies represent the miniaturized endeavors of our times. What’s more, they offer a microcosm of the astonishing progress over the past century in discovering the secrets of life, such as how genetic information is encoded and directs the development of a single cell into the complex architecture of an animals with eyes, legs, wings and all.

People used to set out in ships for voyages to the far side of the world to hunt the sperm whale, source of the clearest light to be had in the days before oil and gas. Nowadays, our greatest explorations are inward, into the molecular workings of cells with ever greater precision and detail. The tools of the

Drosophila researchers use not telescope and harpoon and knife, but microscope and fine forceps and eyelash probe. As with whaling, fruit fly work evokes both grandeur and humility. Some might hardly consider it a respectable occupation. When I was a marine invertebrate biologist, I sneered at model organisms with their platonic designations: the mouse, the worm, the fly. Yet flies have since won me over with their quirky names (of which more to follow), the satisfying algebra of practical genetics, and the strong tradition of collaboration among fly researchers, to name just a few virtues. Many wonders of the deep have been illuminated by the pursuits of the fly researcher.