Molecular mechanisms underlying the prepupal-instar-forming metamorphosis in black soldier fly (Hermetia illucens)

The black soldier fly (Hermetia illucens, Diptera) undergoes an atypical metamorphic program in which a distinct non-feeding prepupal instar precedes pupation, but its developmental and molecular basis remains poorly understood. Here, we investigated this unusual metamorphic program through integrated developmental and RNAi-based analyses. Postembryonic staging confirmed that the 6th instar feeding larva molts into a non-feeding 7th instar prepupa, whose hardened cuticle subsequently serves as the puparium during intra-puparial development. Expression profiles of four key metamorphic genes revealed a stage-specific Chinmo/Kr-h1-Br-c-E93 regulatory shift corresponding to larval, prepupal, and pupal/adult development, with sustained Br-c expression defining the 7th instar prepupal stage. RNAi-mediated knockdown of Kr-h1 and/or Chinmo induced precocious larval-prepupal metamorphosis, supporting their roles in larval stage maintenance, whereas depletion of Br-c or E93 disrupted prepupal-pupal transition and adult differentiation, respectively, consistent with their functions as pupal and adult specifiers. These results support generally conserved functions of the metamorphic gene network while indicating the absence of a repressive effect of Br-c on E93 in H. illucens prepupae. Together, these findings establish the 7th instar prepupa as an independently regulated transitional stage, providing insight into how metamorphic programs are reorganized to diversify life-history strategies in holometabolous insects.