Functional analysis of the wing-selector gene vestigial (vg) in the red flour beetle Tribolium castaneum revealed two groups of vg-dependent tissues in the wingless first thoracic segment (T1) — the carinated margin (a lateral pronotal expansion) and pleural plates (trochantin and epimeron) — that transform into a single ectopic elytron when the Hox gene Sex combs reduced is reduced, providing functional evidence that insect wings arose from both a tergal (paranotal-lobe-like) and a proximal-leg (exite-bearing) component. The authors left open whether these vg-dependent wing-like roles reflect historical serial homology or lineage-specific co-option of the wing gene network, the embryonic fates of the vg-positive domains, the developmental origin of the pleural plates, and the mechanism by which the two outgrowths merge. A decade of follow-up work has broadly confirmed dual tergal-plus-pleural wing-related tissues across cockroaches, beetles, crustaceans, and fossils, but the discriminating questions — homology versus co-option, embryonic fate mapping, and the merger mechanism — remain unresolved, and recent reviews treat the relative contributions of the incorporated exite homolog, pleuron, and tergum to wing evolution as a currently open focus.

Background

Insects are the only arthropods to achieve powered flight, and how the wing arose has been debated for over a century. Two classical hypotheses dominate: the tergal (paranotal) hypothesis, which derives the wing from a lateral expansion of the dorsal body wall (tergum, the thoracic notum), and the pleural (exite) hypothesis, which derives it from an outgrowth (exite) on a proximal leg segment of a crustacean-like ancestor. Insects bear wings on the second and third thoracic segments (T2, T3), while the first thoracic segment (T1) is wingless; in beetles the T2 forewings are hardened into protective elytra (wing covers). Developmental genes identified in Drosophila wing formation include vestigial (vg), a transcriptional cofactor that acts with its DNA-binding partner Scalloped and whose activity confers wing-cell identity and is necessary for wing margin formation and wing primordium induction; apterous (ap), required for dorsal wing fate; Wingless (the Drosophila Wnt1 homolog, a secreted Wnt-family signaling protein); and nubbin (nub), a POU-domain transcription factor used as a wing-lineage marker whose enhancer activity marks future wing tissue. A structure 'serially homologous' to wings is one that shares common evolutionary origin with wings by repetition within the same body plan, as the T3 haltere (the knob-like flight-balancing organ of flies) is serially homologous to the T2 wing. Clark-Hachtel, Linz and Tomoyasu (2013) functionally dissected T1 of the beetle Tribolium castaneum. RNAi depletion (RNA interference, double-stranded-RNA-directed knockdown of a target gene) of vg deleted not only wing and elytron tissue but also specific T1 body-wall structures: the carinated margin (a lateral ridge of the pronotum, the T1 dorsal plate) and two pleural plates — the posterior trochantin and the epimeron — which are lateral body-wall sclerites classically thought to derive from the subcoxa, a proximal leg segment bearing an exite in ancestral arthropods. The carinated margin also required ap and Wingless signaling and showed vg-dependent nubbin enhancer activity, indicating an extended overlap of gene networks with wings. In Cephalothorax (Cx) mutants — allelic combinations of the Hox gene Sex combs reduced (Scr; Hox genes are conserved homeobox transcription factors specifying segmental identity along the body axis), which normally represses wing fate in T1 — an ectopic T1 elytron forms from two distinct outgrowths, one at the carinated margin and one from the epimeron region, which merge into one elytron in strongly transformed individuals. Because the carinated margin parallels the paranotal lobe and the pleural plates parallel the exite-bearing proximal leg segment, the authors proposed that T1 retains two wing serial homologs and that wings arose by a dual origin: merger of a tergal and a pleural component, echoing earlier combinational models of Rasnitsyn and Kukalova-Peck and expression data of Niwa et al. (2010) in basal insects. The authors themselves flagged what their evidence could not settle: vg deployment in nonhomologous tissues could equally reflect deep homology via co-option, or independent loss of vg pleiotropy across lineages; the embryonic vg-positive domains (dorsal tergal-edge ectoderm and characteristic invaginated vg-positive cell clusters at the base of leg primordia in all three thoracic segments) were not fate-mapped to adult structures; the evolutionary and mechanistic origin of the pleural plates (including the requirement for odd-skipped family leg-patterning genes in pleuron development found in their toolkit RNAi screen) required further developmental analysis; the cellular and genetic mechanism of the merger of the two outgrowths remained unexplained; and comparative functional tests in other insects with pronotal expansions, such as hemipteran treehopper helmets, were needed.

Problem Statement

Determine whether the vg-dependent wing-like tissues of the Tribolium first thoracic segment — the carinated margin and the pleural plates (posterior trochantin and epimeron) — are bona fide wing serial homologs reflecting an ancestral dual (tergal plus proximal-leg/exite) wing precursor, or lineage-specific (co-opted or independently retained) deployments of the wing gene network, and thereby establish a robust, phylogenetically supported model of insect wing origin. The source paper flagged several interrelated open questions that remain unresolved and that bear directly on this target: (1) comparative functional evidence across additional insect lineages (and relevant outgroups) discriminating conserved serial homology of the T1 vg-dependent roles from lineage-specific co-option or independent loss of vg pleiotropy; (2) fate-mapping of the Tribolium embryonic vg-positive domains — the dorsal tergal-edge ectoderm and the invaginated vg-positive cell clusters at the leg bases — to the adult carinated margin, pleural plates, and wing primordia; (3) how pleural identity is developmentally and evolutionarily specified, including the mechanistic role of leg-patterning genes such as the odd-skipped family; and (4) a mechanistic account of how the two vg-dependent outgrowths merge into a single ectopic elytron under reduction of the Hox gene Sex combs reduced. These questions all bear on the same inference: whether the wing is an evolutionary merger of two distinct serial homologous tissues. They are mutually reinforcing lines of evidence toward that single determination rather than four independently mandatory deliverables, so the task is resolved by a defensible, phylogenetically supported determination of the homology-versus-co-option alternative that accounts for the evidence available on each line, and no one sub-question pursued in isolation settles the task.

References

  1. Clark-Hachtel CM, Linz DM, Tomoyasu Y (2013) Insights into insect wing origin provided by functional analysis of vestigial in the red flour beetle, Tribolium castaneum. Proceedings of the National Academy of Sciences USA 110(42):16951-16956. DOI:10.1073/pnas.1304332110, https://doi.org/10.1073/pnas.1304332110
  2. Niwa N, Akimoto-Kato A, Niimi T, Tojo K, Machida R, Hayashi S (2010) Evolutionary origin of the insect wing via integration of two developmental modules. Evolution & Development 12(2):168-176. DOI:10.1111/j.1525-142x.2010.00402.x, https://doi.org/10.1111/j.1525-142x.2010.00402.x
  3. Elias-Neto M, Belles X (2016) Tergal and pleural structures contribute to the formation of ectopic prothoracic wings in cockroaches. Royal Society Open Science 3:160347. DOI:10.1098/rsos.160347, https://doi.org/10.1098/rsos.160347
  4. Mashimo Y, Machida R (2017) Embryological evidence substantiates the subcoxal theory on the origin of pleuron in insects. Scientific Reports 7:12597. DOI:10.1038/s41598-017-12728-2, https://doi.org/10.1038/s41598-017-12728-2
  5. Prokop J, Pecharová M, Nel A, Hörnschemeyer T, Krzemińska E, Krzemiński W, Engel MS (2017) Paleozoic Nymphal Wing Pads Support Dual Model of Insect Wing Origins. Current Biology 27:263-269. DOI:10.1016/j.cub.2016.11.021, https://doi.org/10.1016/j.cub.2016.11.021
  6. Clark-Hachtel CM, Moe MR, Tomoyasu Y (2018) Detailed analysis of the prothoracic tissues transforming into wings in the Cephalothorax mutants of the Tribolium beetle. Arthropod Structure & Development 47:352-361. DOI:10.1016/j.asd.2018.06.005, https://doi.org/10.1016/j.asd.2018.06.005
  7. Linz DM, Tomoyasu Y (2018) Dual evolutionary origin of insect wings supported by an investigation of the abdominal wing serial homologs in Tribolium. Proceedings of the National Academy of Sciences USA 115:E658-E667. DOI:10.1073/pnas.1711128115, https://doi.org/10.1073/pnas.1711128115
  8. Hu Y, Linz DM, Moczek AP (2019) Beetle horns evolved from wing serial homologs. Science 366:1004-1007. DOI:10.1126/science.aaw2980, https://doi.org/10.1126/science.aaw2980
  9. Fisher CR, Wegrzyn JL, Jockusch EL (2020) Co-option of wing-patterning genes underlies the evolution of the treehopper helmet. Nature Ecology & Evolution 4:250-260. DOI:10.1038/s41559-019-1054-4, https://doi.org/10.1038/s41559-019-1054-4
  10. Bruce HS, Patel NH (2020) Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments. Nature Ecology & Evolution 4:1703-1712. DOI:10.1038/s41559-020-01349-0, https://doi.org/10.1038/s41559-020-01349-0
  11. Clark-Hachtel CM, Tomoyasu Y (2020) Two sets of candidate crustacean wing homologues and their implication for the origin of insect wings. Nature Ecology & Evolution 4:1694-1702. DOI:10.1038/s41559-020-1257-8, https://doi.org/10.1038/s41559-020-1257-8
  12. Monteiro A (2021) Distinguishing serial homologs from novel traits: Experimental limitations and ideas for improvements. BioEssays 43:e2000162. DOI:10.1002/bies.202000162, https://doi.org/10.1002/bies.202000162
  13. Clark-Hachtel CM, Fernandez-Nicolas A, Belles X, Tomoyasu Y (2021) Tergal and pleural wing-related tissues in the German cockroach and their implication to the evolutionary origin of insect wings. Evolution & Development 23:100-116. DOI:10.1111/ede.12372, https://doi.org/10.1111/ede.12372
  14. Fisher CR, Kratovil JD, Angelini DR, Jockusch EL (2021) Out from under the wing: reconceptualizing the insect wing gene regulatory network as a versatile, general module for body-wall lobes in arthropods. Proceedings of the Royal Society B 288:20211808. DOI:10.1098/rspb.2021.1808, https://doi.org/10.1098/rspb.2021.1808
  15. Hu Y, Moczek AP (2021) Wing serial homologues and the diversification of insect outgrowths: insights from the pupae of scarab beetles. Proceedings of the Royal Society B 288:20202828. DOI:10.1098/rspb.2020.2828, https://doi.org/10.1098/rspb.2020.2828
  16. Tomoyasu Y (2021) What crustaceans can tell us about the evolution of insect wings and other morphologically novel structures. Current Opinion in Genetics & Development 69:48-55. DOI:10.1016/j.gde.2021.02.008, https://doi.org/10.1016/j.gde.2021.02.008
  17. Ohde T, Mito T, Niimi T (2022) A hemimetabolous wing development suggests the wing origin from lateral tergum of a wingless ancestor. Nature Communications 13:979. DOI:10.1038/s41467-022-28624-x, https://doi.org/10.1038/s41467-022-28624-x
  18. Bruce HS, Patel NH (2022) The Daphnia carapace and other novel structures evolved via the cryptic persistence of serial homologs. Current Biology 32:3792-3799. DOI:10.1016/j.cub.2022.06.073, https://doi.org/10.1016/j.cub.2022.06.073
  19. Deem KD, Halfon MS, Tomoyasu Y (2024) A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species. Scientific Reports 14:10078. DOI:10.1038/s41598-024-60432-9, https://doi.org/10.1038/s41598-024-60432-9
  20. Ohde T, Prokop J (2025) The transition to flying insects: lessons from evo-devo and fossils. Current Opinion in Insect Science 68:101332. DOI:10.1016/j.cois.2025.101332, https://doi.org/10.1016/j.cois.2025.101332