Lepidopteran prolegs are novel traits, not leg homologs.

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. A RNA-seq analysis with various appendages of Bicyclus anynana butterfly larvae indicated that the proleg transcriptome resembles the head-horn transcriptome, a novel trait in the lepidoptera, but not a thoracic leg. Under a partial segment abdominal-A (abd-A) knockout, both thoracic leg homologs (pleuropodia) and prolegs developed in the same segment, arguing that both traits are not serial homologs. Further, three of the four coxal marker genes, Sp5, Sp6-9, and araucan, were absent from prolegs, but two endite marker genes, gooseberry and Distal-less, were expressed in prolegs, suggesting that prolegs may be using a modular endite gene-regulatory network (GRN) for their development. We propose that larval prolegs are novel traits derived from the activation of a pre-existing modular endite GRN in the abdomen using abd-A, the same Hox gene that still represses legs in more lateral positions.