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.

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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.

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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.

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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:

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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.