The red flour beetle Tribolium castaneum carries two distinct sets of tissues serially homologous to wings in its wingless body segments, one along the dorsal body wall (tergal) and one in the lateral body wall (pleural), and both merge into a complete ectopic wing when Hox genes are knocked down. This supported the dual-origin hypothesis: insect wings arose from the evolutionary merger of a tergal tissue and a pleural (proximal-leg-derived) tissue. However, which lineage actually builds which part of a real wing — blade, hinge, or articulation — has never been fate-mapped, and it remains unknown whether the two-lineage configuration is ancestral across arthropods or a beetle peculiarity. The problem is to determine quantitatively, by lineage tracing in Tribolium and comparative developmental analysis across taxa, the tergal versus pleural cellular contributions to each wing subregion, whether the pleural lineage preexists or is induced de novo, and thereby to locate the truth on the spectrum of dual-origin scenarios, whose ends range from a purely tergal wing blade to a largely pleural one.

Background

Insects acquired powered flight roughly 400 million years ago, and the evolutionary origin of the wing itself has been debated for over two centuries. Three hypotheses dominate. The tergal (paranotal) hypothesis derives the wing from outgrowths of the dorsal body wall plate (tergum); the pleural (exite) hypothesis derives it from an outgrowth (exite) on the leg base of an ancestral arthropod, a segment of which was incorporated into the lateral body wall (pleuron); and the dual-origin hypothesis, foreshadowed by Crampton (1916) and Rasnitsyn (1981), proposes that both a tergal and a pleural tissue merged to form the wing. Evo-devo studies gave the debate molecular content: vestigial (vg), a wing-specifying gene first characterized in Drosophila, and nubbin (nub), a POU-domain gene marking the wing lineage, are expressed not only in wings but also in specific non-wing structures, which are then called wing serial homologs (structures repeated along the body that share a common developmental origin with wings). Niwa et al. (2010) found modular dual tergal contributions in mayfly and other basal insects, and Clark-Hachtel, Linz and Tomoyasu (2013) showed in the beetle Tribolium castaneum that vg is required both in wings and in a proximal tergal structure, functionally supporting a composite origin. Linz and Tomoyasu (2018) then demonstrated that abdominal segments of Tribolium, which normally bear no appendages, contain two separable wing serial homologs: the pupal defensive gin-traps (a tergal derivative, where Hox genes Ultrabithorax (Ubx) and abdominal-A (abdA) repress wing identity) and a pleural cell population that becomes visible — expressing vg and a nub enhancer-trap reporter (line pu11) — only after Ubx/abdA double RNA interference (RNAi), a method that depletes messenger RNA and thereby removes Hox repression. Upon this Hox knockdown, tergal and pleural cells merge into a complete ectopic wing. This established that a two-lineage configuration exists even outside the wing-bearing segments, and the authors argued it represents the ancestral state of wing serial homologs, supporting a dual evolutionary origin of wings. The study explicitly left open, however, what no experiment has yet delivered: a cellular fate map. The pu11 reporter is also active in some non-wing tissues, the pleural vg-positive population is at or below detection threshold in unmanipulated segments, and it is unknown whether Hox genes directly regulate gin-trap genes or act only indirectly through wing-identity suppression and cell redistribution. Subsequent work sharpened the problem without solving it: Clark-Hachtel and Tomoyasu (2020) found candidate wing homologues in the crustacean Parhyale hawaiensis both in terga and proximal leg segments; Bruce and Patel (2020) argued from Parhyale leg-gene knockouts that wings derive from an ancient exite borne on the precoxal segment (the leg segment adjacent to the coxa, the basal leg segment), which was incorporated dorsally into the body wall (a competing single-lineage interpretation); Clark-Hachtel et al. (2021) confirmed tergal and pleural wing-related tissues in the German cockroach but found additional vg-independent contributions; Ohde et al. (2022) showed cricket wing blades grow from a lateral tergal margin organizer; and Prokop et al. (2017, 2022, 2023) documented Paleozoic nymphs with wing-like lateral outgrowths on thorax and abdomen. Reviews in 2024–2025 (Treidel et al. 2024; Ohde and Prokop 2025) describe the relative contributions of the incorporated exite homolog and surrounding tergal and pleural tissues as the field's current unresolved focus.

Problem Statement

Determine the cellular lineage composition of the insect wing and thereby resolve the spectrum of the dual-origin hypothesis. Specifically: (1) by lineage tracing in Tribolium castaneum — both in normal wing development on the second and third thoracic segments (T2/T3) and in ectopic abdominal wings induced by Ubx/abdA knockdown — fate-map and quantify the contributions of the tergal wing-serial-homolog lineage (including the gin-trap region and dorsal posterior tergal edge) and the pleural wing-serial-homolog lineage to the anatomical subregions of the wing (blade, hinge, and articulatory structures); (2) establish whether the pleural lineage exists as a preexisting vg-positive (or nub-enhancer-positive) cell population below current detection thresholds or is induced de novo upon Hox reduction; and (3) by comparative developmental analysis across a taxonomically broad sample of insects and non-insect arthropods, determine whether the two-lineage (tergal plus pleural) wing-serial-homolog configuration is ancestral or lineage-specific, mapping how the relative tergal-versus-pleural contributions to blade and hinge vary across taxa. All three deliverables above are jointly required for resolution, none being independently sufficient; the comparative sampling in (3) must span more than one insect order together with at least one non-insect arthropod lineage. The single scientific objective uniting these deliverables is a quantitative, evolutionarily grounded account of which ancestral tissues built which parts of the insect wing, locating the truth between the extremes of the dual-origin spectrum (pleural tissue contributing only minor articulation versus forming most of the blade), and adjudicating between the dual-merger and precoxal-exite-incorporation readings of the comparative evidence.

References

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