The stick and leaf insects (Phasmatodea) have modified excretory organs compared to other insects (Ramsay, 1955, Shelomi, 2017, Shelomi and Kimsey, 2014, Shelomi et al., 2015). Their Malpighian tubules, which originate from the midgut/hindgut junction as in other insects, are typically subdivided into two groups (de Sinéty, 1901, Ramsay, 1955, Savage, 1962). The “excretory” or “superior” Malpighian tubules loop anteriorly and then run back, terminating blindly (de Sinéty, 1901, Ramsay, 1955, Shelomi and Kimsey, 2014) near the posterior end of the insect just anterior to the rectum. These tubules perform the ion and nitrogen waste elimination functions typical of Malpighian tubules (Dow, 2009). The “ calciferous” or “inferior” Malpighian tubules do not loop forward, terminate in specific fat body cells called cells of Sidorot (de Sinéty, 1901, Savage, 1962), are filled with a milky fluid, and sequester calcium for later deposition into a biomineralized layer of the eggshells, which is a unique feature to Phasmatodea eggs (Chapman, 1969; Debruyn et al., 2025ab; Farina et al., 2022, Pantel, 1919). The remarkable deposition of either only calcium oxalate (Debruyn et al., 2025a) or a combination of calcium oxalate with calcium carbonate (Debruyn et al., 2025b) into the eggshell provides strong mechanical protection of the embryo (Saltin et al., 2025), impacting the evolution of recent eggshell diversity in Phasmatodea and various functional structures on the eggs (Büscher et al., 2023, Büscher et al., 2024). At least in derived Phasmatodea (Tilgner et al., 1999), these Malpighian tubules appear in groups of five calciferous Malpighian tubules for each excretory Malpighian tubule (Savage, 1962). The number of Malpighian tubules in one insect can surpass 100, obscuring most of the reproductive organs upon dissection (Clark, 1976).

In addition to their Malpighian tubules, Phasmatodea also have another, distinct series of thinner, more contorted tubules that emerge from pyriform [pear-shaped] ampules studding the posterior third of the midgut. These are an autapomorphy of the Phasmatodea (Bartel, 1947, Bradler, 2015, Klass et al., 2002, Ramsay, 1955, Terra et al., 2023, Tilgner et al., 1999) commonly referred to as the midgut “appendages,” “appendices,” “papillae,” or “tubules” (Shelomi et al., 2015). Unlike Malpighian tubules, they are comprised of the same cell type from distal to proximal end (Clark, 1976, Savage, 1962). They number from four in the basally split Timema to several dozen in larger, euphasmatodean species (Shelomi et al., 2015). The motile, tracheated midgut tubules extend into the posterior end of the insect where they end blindly in the hemolymph close to the hindgut between the annulus and sphincter (Ramsay, 1955, Shelomi and Kimsey, 2014, Shelomi et al., 2015). The physiological of the midgut appendages is not understood in detail. Vital staining assays confirm these midgut tubules excrete certain xenobiotic substances while the Malpighian tubules excrete others (Shelomi and Kimsey, 2014, Shelomi et al., 2015). Transcriptomics assays of the midgut tubules found highly expressed transporters for peptides, proteins, phosphatases, lipids, fatty acids, lipoproteins, and fat-soluble vitamins (Shelomi, 2017). They also express carbonic anhydrase activity that increases the midgut pH (Monteiro et al., 2014, Shelomi, 2017).

To gain further insight into the different excretory tubules of the Phasmatodea, new imaging techniques are needed to supplement extant light and electron micrographs and anatomical data (Shelomi and Kimsey, 2014). Here we report on the first use of X-ray micro-computer tomography (micro-CT) on the Phasmatodea abdomen, as has been done in other insects before (De Paula et al., 2022), including specifically to examine the Malpighian tubules (Bell et al., 2012; 2021; Chen et al., 2023, Martín-Vega et al., 2021).