Development of the Pronotal Explanate Margin, a Novel Evolutionary Trait in Tortoise Beetles

Most tortoise beetles, belonging to the subfamily Cassidinae (Coleoptera: Chrysomelidae), possess distinctive explanate margins, comprising elongations of the pronotum and elytra outer margins. These margins flatten against the ground, serving as a unique defensive mechanism against predators. To understand the developmental and evolutionary origins of explanate margins, we examined the development of the pronotal part of these structures in two tortoise beetle species: Thlaspida biramosa (Boheman) (tribe Cassidini) and Laccoptera nepalensis (Boheman) (tribe Aspidimorphini). Although final (fifth) instar larvae of both species exhibited no external prothoracic structures associated with explanate margins, pupae possessed a plate-shaped structure projecting anterolaterally on their pronotum. This plate-shaped structure was identified as the pupal primordium of the pronotal explanate margin, as the explanate margin emerged from inside the structure during eclosion. In prepupae of T. biramosa, the primordial tissue exhibited three region-specific folding and furrowing patterns, beneath larval cuticles. These epithelial structures expanded within minutes at the onset of pupation, resulting in pupal primordial formation. Thus, pronotal explanate margins originate from pronotal epithelia, with the furrowing and folding patterns of the epithelia shaping pupal and even adult pronotal explanate margins. The presence of morphologically similar pupal pronotal projections in various Cassidinae suggests that the development of pronotal explanate margins is likely common in these beetles.