I’m Off to Grad School!

A Caterpillar’s Life After Death

While I have written in the past about my interest in plant cell culture, my current project is with animals–an even more difficult group of cells to culture. Luckily, insect cells are much less finicky than mammalian cells, and methods have been established and refined since Thomas Grace at CSIRO first reported culturing cells from Opodiphthera eucalypti in 1962. During the ensuing five decades, advances were made in improving Grace’s media with endless variations, developing new formulas for sera-free defined media, and studying the effects of hormones, antibiotics, and additives on cell growth, differentiation, and protein synthesis. In addition, new protocols have been published for initiating cell lines from a growing breadth of terrestrial and aquatic arthropods and from a longer list of tissues, and for enlisting permanent cell lines in the production of insecticidal baculoviruses and transgenic proteins.

Despite all these advances, insect cell cultures are still Lepidoptera-centric, partly because many species are economically significant, well-characterized agricultural pests, and partly because they possess relatively large and easily reared larvae. These two attributes are especially apt in describing the tobacco hornworm (Manduca sexta), which for these very reasons has emerged as one of the de facto models for physiological, developmental, ecological, and behavioral studies of the insects as a whole and the Lepidoptera in particular. Although it thrives outdoors on solanaceous plants to the chagrin of home gardeners and tobacco farmers throughout the United States, captive populations maintained by scientists have readily adapted to crowded, sterile conditions on artificial diets. Larvae mature in just 2 or 3 weeks at a weight of 11 grams or more, allowing experiments to be conducted at nearly the same scale as would be for an adult lab mouse. And with the Manduca draft genome released just this August, we can expect many more insights from this big green worm.

A pinned hornworm, ready to eat prepared for tissue isolation. (After this dissection I learned that lateral cuts are not ideal when finding small organs like testes and imaginal discs.)

But one way in which I have not seen Manduca fully utilized is in cell line development. Instead, the current focus in lepidopteran cell line research is less in mining various species for promising new lines and more in the development of protocols for transfecting insect cells to produce harvestable recombinant proteins for further study and pharmaceutical use. For these purposes, already well-entrenched cell lines like Sf9 from Spodoptera frugiperda and High-Five from Trichoplusia ni already suffice. M. sexta cell lines exist, but are limited to those from eggs and hatchling larvae. What I set out to accomplish with my project, then, was to start primary cell cultures from mature 5th instar Manduca larvae, with the overly-optimistic hope that one of these would later on establish a permanent cell line.

Internal anatomy may intimidate novices to insect dissection, but in fact it is quite simple once one knows what to look for. In this caterpillar, a dorsal incision was made from T1 (the first thoracic segment, just behind the head) to the end of A5 (the fifth abdominal segment), and the flaps pinned down to expose the body cavity. The wide yellow digestive tract is the most obvious feature, and is supplied with a network of webby, silvery tracheoles that transport oxygen and carbon dioxide. The white fluff lining the body cavity is the peripheral fat body, which stores fats and glycogens. The testes are clear bubbles lying on either side of the dorsal aorta, a tubular muscle that circulates the hemolymph. A difficult detail to make out is a yellowish thread, the distal portion of a single Malpighian tubule, involved in excretion and ion balance.

My target organs are larval midgut and fat body, which both contain their own stem cells, plus testes and wing imaginal discs that have a clear potential for growth as incipient adult structures. Last week, I dissected these organs and collected them in droplets of media, which I then held in sterile flasks and shut in my lab desk drawer for the weekend. Since this was all an exploratory study to prepare myself to compose a protocol and guide a class of students through it, I did not expect this to actually work.

The four wing imaginal discs in caterpillars are internal wing buds retained throughout the larval stage that rapidly expand in size prior to pupation. In Manduca, they are attached to the exoskeleton and are visible externally as small white blemishes on the 2nd and 3rd thoracic segments, in line with the row of spiracles (breathing holes) running down the other body segments. Image by Derek Hennen.

But five days later:


The first picture shows a culture of numerous, very small cells having just migrated from an imaginal disc; the second picture directly above shows larger, more spread out cells from a fat body culture. Both cell types have adhered directly to the plastic; the organs themselves also attached over the last few days. Interestingly, while the fat bodies are still intact, the discs have completely disintegrated, leaving nothing but a naked tracheole framework and a puddle of small round cells busily secreting matrix. On the other hand, the fat body cells have assumed a fibroblast-like morphology, with a single clear organelle (possibly a nucleus?) visible. These were the only viable cell cultures thus far; the testes and midguts have not yet shown any activity so far.

I’m incredibly excited at the ease with which I was able to initiate these primary cultures, and it gives me hope that my fellow students will share in these beautiful results when I lead them through this experiment in a few weeks. After all, there is something exciting about taking cells from a complex, multicellular animal, then growing those cells in a flask indefinitely. In a way, it’s another way to keep the caterpillars that I’ve always been so fond of: Rather than nurturing individuals from the moment they are laid as eggs to their final shuddering wingbeats as worn, dying adults, I can remove their cells from that inevitable cycle of life and death and fix them in a state of eternal, fecund youth, stuck to the side of a plastic microcosm and swimming forever in nutrient-rich ambrosia. And on a moral level, I feel slightly absolved of my guilt of killing these magnificent animals for their cells: In a way, while their lives have been terminated, I’m giving their cells a shot at an immortal afterlife.

How Callus

Since graduation, I’ve been on a bit of a plant kick–not just because I needed to learn plant physiology and ecology for my GRE subject test and for my exploration of the flora and fauna near my new home near a suburban conservation area–but also because of my newfound appreciation for horticulture as art and discipline distinct from the botanical and ecological sciences, and for the therapeutic effect that gardening has had for my recent depression. As a side to my experimentation with tropical hibiscus propagation this summer, I was recently able to use my hibiscus calli in class in addition to the plant roots required for the day’s lab protocol. We performed assays to demonstrate areas of high enzymatic activity, and therefore identify zones of rapid cell growth and division, in these plant tissues. One of these was an assay for peroxidase, which neutralizes reactive oxygen species generated by metabolism that can destroy cell machinery and DNA.

Callus cells scraped off a hibiscus stem look like clear, sausage-shaped bubbles. 9/20/2016.

Callus tissues are essentially stem cells in the somatic tissues of land plants. These stem cells are derived from more specialized cells that undergo rapid growth and division in the presence of growth hormones in the correct proportion. These callus form disorganized masses before differentiating into new roots and shoots, depending again on the appropriate mixture of hormones. In nature, callus tissue is encountered in plant wounds, where they grow and divide rapidly to heal over the injury. Calli are most frequently studied in vitro, however, since they are commonly used in plant tissue culture to produce numerous clones of a desired plant from single mature cells. In such situations, calli are the plant analogs of bacterial colonies in agar dishes.

Exposed to peroxidase substrate, the cells’ endogenous peroxidases break down the substrate, producing a purple product. 10/11/2016.

I personally find it fascinating that such tissues can be generated on a hibiscus cutting in a jar of water, without sterile technique or specialized tools or media. Someday, I’d like to see if such in vivo callus cells are capable of forming plant embryos in culture, without having to induce calli in vitro. In fact, hibiscus cuttings are quite unique in how they develop such extensive masses of callus tissue all along the submerged part of the stem, not just in wounds. This disorganized tissue differentiates into functional roots in time, even without rooting hormone. This propensity to produce callus prior to rooting makes tropical hibiscus as a group rather easy to propagate, a trait that has made them so amenable to cultivation by enamored enthusiasts around the world. Soft green stem cuttings, taken any time of year, are relatively easy to root in water or soil, as long as adequate warmth and high humidity are provided.


My Wild Adventures With (Rooting) Datura Stem Cuttings

Taking a walk in Allegheny Commons a month ago, I noticed a weedy looking shrub peeking out amid the cannas and elephant ears flopping lazily under the wilting sun. Weedy, yes, but somehow it managed to fit in with its tropical ornamental neighbors. Its succulent purple stems and erect but bushy habit were attractive enough to catch my attention even next to the taller and flashier cannas. Velvety, dark green, heart-shaped leaves sheltered spiny seedpods nodding on thick stems from the forking shoot tips, like botanical maces melting away à la The Persistence of Memory. But these unusual features formed merely the backdrop to the huge, striking white blossoms, their scroll-shaped buds flushing a slight lavender as they unfurled into the late afternoon sky.

The appearance, orientation, and ripening of the seedpods are important characters to pay attention to when identifying Datura. 10/13/2016.

Anyone with an eye for gardening would recognize these exotic features anywhere on the planet as belonging to the plants of the genus Datura. Their common names are much more descriptive: jimsonweeds, thornapples, moonflowers, angel’s trumpets. Although their taxonomic history is rather complicated, I later tentatively identified this particular species as D. inoxia subsp. quinquecuspida, based on this old-ish online key from Erowid, adapted from one by Richard Sanders at the American Brugmansia and  Datura Society (designated as the authority on Datura taxonomy by the ICNCP in 2004).

Datura inoxia in Allegheny Commons, 10/13/2016. The opened flowers resemble flaring trumpet horns, hence the Chinese name, la ba hua (trumpet flower). With the ribbing and the thin bending petiole, I think they look more like an old-fashioned grammophone horn.

Datura‘s seductive pure white flowers and weedy abundance in out-of-the-way areas stand in eerily stark contrast to its storied reputation as an unpleasantly hallucinogenic drug and a deadly poison. I’ve seen Datura bushes in front of scruffy old homes in college neighborhoods (!) and cultivated in a row in front of suburban apartments filled with retirees (!!). Tom Scocca at the old Gawker website has a great essay in which he stumbles upon a Datura sprouting innocuously from a patch of bare dirt in the middle of New York City, blooming and reseeding and as wholly ignorant of its own toxic potential as all the daily passersby occupied with their own duties to grow and reproduce. Indeed, every afternoon when I would pick several D. inoxia flower buds to bloom at home, I would get strange looks from the dog walkers and outbound commuters who could not comprehend why I was so intently stomping around in an overgrown garden in an urban center.* But for me, the plant’s treacherous snake-in-the-grass character only augments the rare combination of grace and hardiness that convinced me that I had to have one of my own. Truly, for those who grow Datura on purpose (whatever purpose that might be), this is a plant that not only sits and looks pretty on its own, but has a dangerous reputation whose story adds to its mysterious, literally intoxicating charisma.


I took two D. inoxia cuttings on September 23 in the late afternoon, the worst season and time of day to take cuttings for most plants. Even worse, these were tip cuttings on a mature and blooming plant. For whatever reason (hmmm) there are not many definitive online guides on how to propagate Datura compared to its less infamous showy relative Brugmansia; many sources recommended propagation by roots or from seed, and only described stem cutting propagation for Brugmansia. However, these were stems I had used to feed my Manduca sexta hornworm caterpillar; after it died I only kept them around because I was too lazy to get rid of them. The whole time they sat neglected on my dresser, they remained succulent and green. The only change seemed to be the development of numerous small white bumps along the submerged sections of the stems. Meanwhile, the cutting wound appeared to turn brown and deteriorate.

Two weeks later, on October 6, I had to tidy up my room and noticed the first short root coming down out of the seemingly rotting wound on one cutting. By Saturday, October 8, that root had doubled in size and many more white tips were poking out of the wound; the other cutting had begun to root as well.

In retrospect, my success is not that surprising considering how related solanaceous plants are also commonly propagated by stem cuttings, like tomato plants and the aforementioned allied Brugmansia. In fact, I wonder if propensity to root by certain cutting techniques might be a way to differentiate similar populations or species. Certainly propensity to root is selected for under cultivation and some lines or varieties have higher success rates than others, so it probably has a genetic basis. It might even be a character state that develops and reverses in plant lineages; certain species might become better at rooting, like aquatic plants and succulents, or worse at rooting,like woody plants, as part of an adaptive syndrome to life in certain environments.

But TL;DR, here is how I would now recommend propagating Datura inoxia from stem cuttings:

  1. Get yourself some shoots from a flowering plant in late summer.
  2. Put them in water; leave alone for some weeks. No rooting hormone or any other cheats needed.
  3. When you’re done, now plant in suitable fast drying, low organic content soil and hope it overwinters in your house.

Not really complicated stuff, even if you’ve been hallucinating nightmares, your eyes are maximally dilated and you’re severely dehydrated, nauseous, dizzy, and just starting to separate dream from reality.

With the cold setting in rapidly, the aboveground portions of the Datura will be killed by frost in the coming weeks. But they’ll be back…


*One morning at the park, I did have an interesting conversation with a nice old lady who was picking the unripened alkaloid-laden fruits, which she claimed to extract in oil as a topical application for her back and leg pains. Hmm, OK, I said.

A Poem About Biological Systematics

Biological systematics is, let’s be honest… not a very sexy field of biology for most people. There is certainly something almost noble in the century-long quest to discover and classify all life on Earth and to unravel the deep and entangled thickets of the tree of life, to understand the literal origins of species. Not many scientific disciplines, I think, can boast such an inherent adventurousness. But that is all buried under layers of technical hemming and hawing about methods of tree construction, character validity, and taxonomic interpretation, all expressed in a jargon peculiar the the field, even within biology. So it’s not often that one sees systematics appear in the writings of non-scientists. But I did recently stumble on a poem, The Rose Family, by Robert Frost (1928):

The rose is a rose,
And was always a rose.
But the theory now goes
That the apple’s a rose,
And the pear is, and so’s
The plum, I suppose.
The dear only knows
What will next prove a rose.
You, of course, are a rose —
But were always a rose.

The poem lightly pokes fun at the seemingly pinheaded idea of apples, pears, and plums being classified in the same family, Rosaceae as regular roses. I’m sure Frost would have loved to know that blackberries and strawberries are in the Rosaceae as well; in fact, we now know that these two groups are even closer relatives of roses than Frost’s fruit trees.

A quince tree (Cydonia oblonga); or, as we now know, a very tasty rose. From Wikipedia.

Of course, the scientific foundation on which this poem rests is logically shaky (rose relatives are emphatically not roses, the same way your cousin, your relative, is not you) but the point is to explain one’s love as constant and steadfast in its devotion even as others’ attentions, tastes, and attitudes wander unpredictably over time. The use of the romantic connotation of flowers, in particular of the rose as opposed to less well-regarded blossoms, deepens the poem’s intentions. It’s a quintessential Frost poem: short, intense, and sweet, but executed with a light touch, like the fragrance of a rose.


(Featured image: An assortment of what most would call hawthorn (Crataegus), but are they actually roses?? From Wikipedia)

My First Animation

I spent a couple spare hours tonight fiddling with Animation Desk, a free basic drawing/animation app I found in the Microsoft app store. It’s really quite addictive, being flexible and easy to use even for those of us who have no art skills. I was so amused with my first effort that I just had to post it here:

(There are 12 frames in this video playing at 24 frames/second, making the whole thing only half a second long; if you play the video on Youtube you can change the settings in the lower right corner of the video to play at slower speed, or right click on the video to loop.)

I really got into animated television shows with Avatar: The Last Airbender in freshman year of college, and from there progressed onto anime as well as American-produced series. Animation has a reputation (in the U.S. at least) of being “for kids”, which often precludes them from being taken seriously by critics as works of art with messages as substantive as their live-action counterparts. If anything, one great advantage of animation is its enormous potential to transcend what is “real”, freeing an artist to exaggerate action, expressiveness, and dialogue as well as create worlds in which viewers can suspend all disbelief. Imagine Kill la Kill, a wild show with transforming school uniforms that examines how our clothes shape our identities and roles, without its schizophrenic transformation sequences and brilliant colors. Or what about The Venture Bros., a self-aware parody of popular science fiction and fantasy tropes, with its elaborate blunders and baroque machinations orchestrated not by its zany animated cast, but by live actors? And even for shows like Spongebob Squarepants, animation facilitates comedy by exaggerating facial expressions, distorting physics, and setting up scenarios whose impossibility in our world renders them all the more entertaining.

And lastly, animation is democratic. A complete, coherent episode may take skilled staff months to complete, but even so, any schmuck can, with a bit of time, stitch together a sequence of doodles into moving, living artwork. Hence this clip: 12 frames of a stick figure flying around with explosions, nothing compared to a complete episode or even a scene, but something I’m kind of proud of nonetheless.

Flies in Winter

This winter has been unusually mild, with snowfall not accumulating until late December and temperatures abnormally warm except for a few freezes in my area. There was one single huge snowstorm just a week ago that buried the northeast and the south, but that bypassed my area and was over quickly, even in places like NYC and Washington, DC that were hardest hit. And these past few days, we have been in the midst of a heat wave, with daytime temperatures reaching the 60s. Not so bad for mid-winter.

Because it was so sunny and warm yesterday, I decided to take some of the houseplants out in the backyard for some fresh air n’ photosynthesis. There, I made my first interesting natural history discovery of 2016.


While moving my pelargoniums from the lawn onto the back porch, I noticed a pale insect huddling in the litter in the pot. This was a newly emerged fly, seen in the minutes after it emerged from its puparium, with its wings recently expanded but its cuticle still soft and unpigmented. How it ended up in the pot isn’t clear; I’m thinking it emerged from the lawn and climbed up and into the pot, rather than remaining dormant in the pot in my house throughout the winter. And these insects are relatively difficult to find in this state: many flies emerge, expand their wings, harden, and are ready for liftoff in as little as an hour.

You’ll notice this fly has a weird snout on its face. That snout is the remains of a structure called a ptilinum, a soft, bladderlike structure between the eyes which the fly repeatedly inflates with blood during its emergence. The puparium, the cramped outer shell that surrounds a fly pupa during its development, can be extremely tough to break from the inside, especially for an insect that has no biting mouthparts. But by pumping blood into its head and expanding the ptilinum, the fly uses a hydraulically powered battering ram to literally push the top off of the puparium and escape its time capsule. In some parasitic flies, the ptilinum is even used to break out of the earthen nests made by their bee hosts:

Given its importance in a brief but crucial time in the fly life cycle, the ptilinum is a pretty sophisticated structure, with associated musculature and sensory hairs. But once the fly has escaped, the ptilinum, now useless, begins to retract into the head. Over the next few days, the cuticle of the ptilinum dissolves, its muscles disappear, and all that is left externally is a thin line (the ptilinal suture) that marks the site where the ptilinum has shrunken.

From this angle you can see the shrinking ptilinum from the side. The rounded tip of the abdomen indicates this fly is male. I wondered what the orange spot was–I’m guessing it’s a testis, since some male caterpillars can be sexed based on the presence of testes showing through the thin cuticle as faint yellow spots.

Over the next thirty minutes, the fly colored up considerably. The stripes on the thorax indicate this fly is a flesh fly, in the large genus Sarcophaga. From these photos alone it is virtually impossible to pin the ID down to species.


That afternoon I saw two other flies buzzing around outside; one came to settle on a plant dish for some water. Indeed, flesh flies, along with blow flies like Pollenia and Calliphora, are some of the earliest ‘large’ insects to appear outdoors, and many flies that overwinter as adults will come out briefly during warm sunny days and retreat when the cold returns. But I don’t know if it’s normal for flies like this one to break out of diapause in the middle of winter, or whether these callow individuals survive until spring without a chance to feed. But who knows whether this behavior is normal at all, what with the eerily uneventful winter we have this year?