Calcium storage in Malpighian tubules and the putative use for pupal chamber formation in a wood-feeding insect

Cerambycid beetles form a chamber to spend their pupal stages in various forms according to the species. The red-necked longhorn beetle Aromia bungii (Coleoptera: Cerambycidae), which is an invasive pest that severely damages Rosaceae trees, makes a pupal chamber at the end of a tunnel deep in the xylem. Beetle larvae and the closely related species form a calcareous lid at the entrance of a pupal chamber. Previous studies on the closely related species conducted more than a century ago suggested that Malpighian tubules (MTs) play a vital role in calcium carbonate accumulation. However, the association between this Ca²⁺ accumulation and pupal chamber lid formation utilizing the possible calcium compounds stored in MTs have not yet been demonstrated. First, we artificially reared A. bungii larvae from eggs in host branches for 100 days and identified the larval developmental status and pupal chamber formation, using X-ray computed tomography. Second, we collected larvae from the branches and observed the internal organs by direct dissection under a microscope. Finally, we analyzed the elemental distribution, particularly calcium, in the larval gut with MTs, using energy dispersive X-ray fluorescence. The results suggest that immature larvae of A. bungii can accumulate Ca²⁺ in the MTs through wood tunneling and feeding activities. Ca²⁺ was stored at the proximal regions in two of the six MTs located posteriorly in the body. Additionally, larvae that formed a calcareous lid at the entrance of pupal chambers in the branches did not store Ca²⁺ in the MTs, suggesting that the larvae of A. bungii used the stored Ca²⁺ in their MTs for lid formation.