Sequencing the genome of the mayfly Cloeon dipterum — a member of Ephemeroptera, the sister lineage of nearly all other winged insects — revealed transcriptomic similarity between nymphal abdominal gills and wing pads, a deeply conserved core of wing-associated genes, the largest odorant-binding-protein (OBP) gene repertoire then known, and a sexually dimorphic opsin expansion in male eyes, while leaving open which evolutionary scenario these findings support. The source paper itself posed the following unresolved questions: (i) whether the gill–wing similarity reflects genuine structural homology (pleural or dual wing-origin scenarios) or convergent gene co-option, i.e. the two-century-old problem of the evolutionary origin of insect wings; (ii) whether the conserved ~130-gene wing core extends to additional pterygote lineages and developmental stages, and which of its genes may have been independently recruited to wing development; (iii) whether gill OBP-expressing cells are chemosensory and what roles the expanded OBP genes play; and (iv) what behavioral role the expanded, sexually dimorphic UV and blue opsins play in mating.

Background

Insects are the only invertebrates to have evolved powered flight, and the acquisition of wings on the second and third thoracic segments was a decisive step in their radiation. Where those wings came from has been debated for over a century. Three families of hypotheses dominate. In the tergal (or paranotal) hypothesis, wings arose as fixed lateral expansions of the dorsal body wall (tergum). In the pleural hypothesis, wings derive from an exite — an outer branch of an ancestral leg — that became incorporated into the lateral body wall (pleuron) of the thorax; the flattened abdominal tracheal gills that aquatic mayfly nymphs use for gas exchange are candidate surviving homologues of such proximal leg exites. In the dual hypothesis, wings assembled from both a tergal and a pleural/exite contribution. Mayflies (order Ephemeroptera), together with dragonflies, form Paleoptera, the sister group of all other winged insects (Neoptera), and their nymphs are aquatic — a combination that makes them a key comparator for testing these scenarios. Almudi et al. (2020) sequenced the genome of the mayfly Cloeon dipterum and profiled gene expression across its life cycle and organs. Comparing co-expression modules (groups of genes whose expression covaries across samples) between mayfly and Drosophila, they found that the mayfly wing-pad module correlated most strongly with the Drosophila wing module, sharing 126 orthologous gene families (130 and 128 genes in Drosophila and Cloeon respectively); 96 of the 130 had no previously described wing function, yet RNA interference against eight of them in Drosophila produced wing phenotypes in every case. Strikingly, the transcriptome of nymphal abdominal gills also resembled that of wing pads, a result consistent with both the pleural scenario (wings as serial homologues of gills) and the dual scenario — but, as the authors stressed, equally consistent with gene co-option, i.e. the same genes being independently recruited into two organs that share epithelial folding and venation-like programs without being homologous. The same study found 191 OBP genes — the largest repertoire of these small soluble odorant-shuttling proteins then described (the previous record, a cockroach, had 109) — with 82 of the 167 complete-model OBPs expressed in gills, 25 of them gill-specific; in situ hybridization placed several in discrete gill cell clusters adjacent to neural markers, suggesting an undescribed sensory role for gills. Adults, meanwhile, use an enlarged opsin set (opsins are the light-sensitive G-protein-coupled receptors of photoreceptors) in a sexually dimorphic way: males of Baetid mayflies carry an extra dorsal pair of compound eyes ('turbanate eyes'), and specific UV (Ops4) and blue (Ops2) opsins are expressed only there, hinting at a mating-related visual function in the twilight swarms that punctuate mayflies' hours-long adult lives.

Problem Statement

The problem, as posed by the Cloeon dipterum genome analysis, has one overarching objective — to explain, from the mayfly system, how insect wings originated and how a basal winged insect encodes its aquatic and aerial adaptations — decomposed into the following source-faithful open questions: (1) Determine whether the transcriptomic similarity between mayfly nymphal abdominal gills and thoracic wing pads reflects true structural homology — i.e. whether wings descend from gill-like pleural exites (pleural scenario) or from a tergal-plus-pleural fusion (dual scenario) — or instead from convergent gene co-option between non-homologous organs; an answer must discriminate among these explanations — on evidence of shared developmental origin or regulatory control, not co-expression alone — rather than merely add further similarities. (2) Refine the conserved core of wing-associated genes (126 orthologous families; 130/128 genes in Drosophila and Cloeon) through studies in additional pterygote lineages and developmental stages (pterygotes, the winged insects), and identify potential cases of genes that could have been independently recruited (co-opted) to wing development in mayflies and Drosophila. (3) Determine whether the OBP-expressing, neural-marker-associated cells in mayfly gills have a chemosensory function — the novel sensory role proposed for gills — and clarify the roles of the expanded OBP genes (191 genes in Cloeon), which remain unclear. (4) Determine the behavioural significance, if any, of the expanded UV- and blue-opsin repertoire and of the male-specific turbanate-eye opsins (UV-Ops4, blue-Ops2) in mayfly mating. Components (1)–(2) form the coupled wing-origin core; components (3)–(4) are the sensory-adaptation components. A solution must address the coupled wing-origin core (1)–(2); components (3)–(4) are separately addressable open directions that the source offers as foundations for future exploration and may be addressed independently in addition.

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