Many years ago in Copenhagen, future Nobel Prize winner August Krogh did what he did best: study a peculiar animal for a very particular reason. He focused on the gas-filled sacs of Chaoborus midges, which he found curiously reminiscent of fish swim bladders. Through a series of experiments, Krogh demonstrated that the midges used the sacs as ballasts, expanding them to float and contracting them to sink, but he couldn't figure out how they accomplished this feat and eventually moved on to other interests. Now published in Current Biology, graduate student Evan McKenzie of the University of British Columbia and colleagues solve Krogh's century old mystery, revealing that it all came down to a common insect protein used in a unique way.
Chaoborus air sacs are unusual: they are completely cut off from the respiratory system and enclosed by a wall made of alternating bands of chitin, a hard polymer found in insect shells, and an unidentified protein. Curious about these mysterious bands and their potential role in regulating buoyancy, the authors examined the sacs under a microscope. Surprisingly, the sacs glowed bright blue under UV light. As zoologists well-versed in the idiosyncrasies of insects, they knew that this could only mean one thing: the unknown protein was resilin; a stretchy, biological rubber found in the elastic tendons of locusts, fleas and other arthropods. An unusual quirk of resilin is that it swells under certain conditions such as high pH. The scientists reasoned that reversible swelling or shrivelling of the resilin bands within the air sac wall could alter the sac's volume and, therefore, adjust the midge's buoyancy. They tested this idea by exposing freshly dissected air sacs to acidic or basic conditions, finding that the air sacs shrunk up to 20% in acid and expanded by 45% in base, confirming their hunch.
The midges only have to change the pH around their air sacs to float or to sink. Like many organisms, insects control the pH of their guts, mitochondria and other compartments with specialized proteins called proton pumps that move hydrogen ions across cell membranes. The researchers tested if these ubiquitous proteins also controlled the pH of the air sac by exposing them to drugs that directly or indirectly interfered with proton pumps. When the team did so, the sacs expanded like they did in basic conditions, confirming that pH controlled the air sac volume and that a specific type of proton pump called vacuolar-type H+ ATPase controlled the pH.
Other insect tissues turn this proton pumping ATPase on or off using cellular signalling pathways involving compounds like cyclic AMP or cyclic GMP. To find out whether the midges use either of these pathways to regulate their buoyancy, the researchers exposed dissected air sacs to synthetic versions of AMP or GMP, each attuned to different branches of the signalling pathway, and observed which compounds caused the sacs to expand. This experiment revealed not only that cyclic AMP co-ordinated the expansion of the air sac, but also identified two more classes of proteins, protein kinase A and cyclic AMP-activated exchange proteins, which are involved in the pH-control signalling pathway, setting the foundation for deeper dives into the unusual physiology of these insects.
Chaoborus midges are the only planktonic insects – in fact the only non-fish – that regulate their buoyancy using a gas bladder and they are the only organism to use resilin as a pH-activated means of motion. Unique on three counts, it is perhaps not surprising that they caught the attention of August Krogh or that it took scientists over 100 years to understand how their unique organ works.