A leaf insect, Phyllium siccifolium. Photo credit: Matan Shelomi
A leaf insect, Phyllium siccifolium. Photo credit: Matan Shelomi

One would think that scientists had already figured out everything there is to know about animal anatomy, considering how long we have been dissecting and describing. However, several mysteries from the past remain unsolved, either because they were forgotten or because more advanced methods were needed to figure them out.

One such puzzle involved the “appendices of the midgut,” a unique set of thin, hollow tubules about the diameter of a human hair that attach to tiny bulbs opening into the digestive tracts of stick and leaf insects, in the order Phasmatodea. The nearly 3000 species of Phasmatodea are famous masters of camouflage, blending into the plants they spend their whole lives eating. The appendices are much less familiar: first described in the 1890s, their functions were completely unknown, as none of the other 1 million species of insects have them. They don’t even have a proper name, as “appendices” unfairly suggests they are vestigial, when instead more derived stick insects have more and more appendices.

The only similar organs are the Malpighian tubules that function as the insects’ kidneys, eliminating waste from the body into the gut for elimination. This was discovered in the 1950’s by a scientist named J. A. Ramsay, who cleverly dissected them into droplets of solutions similar to insect blood and measured what comes out of the other end. Ironically, he did this research using stick insect Malpighian tubules, yet, while aware of the existence of the appendices, he never used his assay to study them. Perhaps they were too thin, or too difficult to work with. The hypothesis that the appendices are modified Malpighian tubules remained untested.

The mystery of what the stick insect appendices actually do lay dormant for several decades, until a few studies in the 21st century revived interest. Two studies noted that the pH of the insect midgut turns from acidic to neutral or even basic at the moment the appendices first appear. One team hypothesized they produce an enzyme called carbonic anhydrase, another that they absorb basic compounds from the insect’s body cavity and excrete them into the gut. Another study injected different colored dyes into living stick insects and dissected them to see which organs absorbed the dyes: the Malpighian tubules or the appendices. They found that some dyes were selectively picked up by one or the other organ, suggesting excretion in the stick insect had been divided among the organs, yet an alternative explanation is that the dyes were binding to something else in the appendices. The basic biological and chemical methods had revealed all they can.

Close-up of the appendices of the midgut: organs only found in stick insects whose functions are still partly unknown. Photo credit: Matan Shelomi
Close-up of the appendices of the midgut: organs only found in stick insects whose functions are still partly unknown. Photo credit: Matan Shelomi

Enter a molecular biology technique called transcriptomics. Whereas genomics reveals all the genes of an organism, which are the same for almost all cells in an individual, transcriptomics shows only the genes being expressed into proteins in the specific tissues or even cells at the exact moment they were sampled. A transcriptomics comparison between the appendices, Malpighian tubules, and the gut could thus indirectly reveal their different functions where biochemical assays could not. As a bonus, because the transcriptome covers every gene being transcribed, it can identify functions that nobody had thought to look for.

That was exactly what happened when the first transcriptomics assay of the stick insect excretory organs was carried out. As expected, the appendices express high levels of carbonic anhydrase: that hypothesis was true. They also expressed different transporter proteins compared to the stick insect Malpighian tubules: transporters that in other insects are still in the latter. Thus the hypothesis that excretory functions were split among the organs was true. What could not have been predicted was that the appendices also express a large number of fat metabolism, transport, and storage proteins. That was a complete surprise: no other insect has fat-storing tubes of any kind. Later Phasmatodea transcriptome studies revealed an absence of lipiphorins: fat-transporting proteins found in most other insects. A new hypothesis had been generated: the appendices are involved in transporting fats or lipophilic substances. Biochemical confirmation of this hypothesis still needs to be done.

This is only one of the surprises Phasmatodea transcriptomics have revealed. These well-hidden creatures were hiding several secrets of their own. Transcriptomics is a powerful tool that can not only solve century old physiological mysteries, but also lead to discoveries people didn’t even think to look for.

Transcriptomics in Entomological Research

A new book on “Transcriptomics in Entomological Research” is now available to introduce this tool to scientists interested in learning more.


  1. dugujdb3 on 21st May 2020 at 5:50 pm

    wow so cool

  2. Stefan Groothuis on 5th April 2022 at 8:05 am

    i agree with this guy ^

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