Mayflies (Ephemeroptera) are the only extant insects that molt after forming functional wings, passing through a winged subimago stage before the adult. Kamsoi, Ventos-Alfonso, Casares, Almudi and Belles (2021) showed in Cloeon dipterum that methoprene (a juvenile hormone mimic) applied at the last nymphal instar induces Krüppel homolog 1 (Kr-h1), represses E93, and blocks subimago formation, producing a supernumerary nymph — consistent with the MEKRE93 metamorphosis pathway known from neopteran insects and implying the subimago is a first instar of the adult stage. However, the causal Kr-h1–E93 interaction could not be validated: dsRNA injections failed to deplete either transcript. No loss-of-function genetics exists for any mayfly species to date, whereas the sister paleopteran order Odonata achieved full RNAi validation of the same pathway in 2022 (Okude et al., 2022). The open problem comprises the source paper's five linked issues: loss-of-function validation of Kr-h1/E93 in Ephemeroptera; completion of the Broad-complex (Br-C) isoform inventory (three zinc-finger isoforms found, others not excluded); decomposing the adaptive significance of the subimago (distributed growth of adult structures versus water-repellent (hydrofuge) cuticle easing the water–air transition); the endocrine basis of the low juvenile-hormone sensitivity (methoprene effective only at 50 µg, not at 0.1–5 µg); and comparative study of the lability of central-filament loss and hook-shaped distal tarsomere formation across Ephemeroptera.

Background

Insects molt throughout juvenile life, but among winged insects (Pterygota) only the mayflies (Ephemeroptera, together with dragonflies the 'Paleoptera', the earliest-branching winged lineages) continue molting after forming functional wings: the last nymphal instar ecdyses to a winged subimago, which molts once more to the adult (imago). Whether the subimago is a modified nymph, a kind of pre-adult, or a pupa-equivalent stage has been debated for over a century, as has the question of why mayflies retain a stage that requires molting fragile membranous wings. Metamorphosis in the better-studied neopteran insects (all winged insects except mayflies and dragonflies) is governed by two hormones — juvenile hormone (JH), which represses metamorphosis, and 20-hydroxyecdysone (20E), which drives molts — acting through a transcription-factor cassette termed the MEKRE93 pathway: JH, via its receptor Methoprene-tolerant (Met), induces Krüppel homolog 1 (Kr-h1, a zinc-finger transcription factor and universal anti-metamorphic factor), which directly represses E93 (an ecdysone-induced helix-turn-helix transcription factor that promotes adult differentiation); a fourth factor, Broad-complex (Br-C, a BTB/zinc-finger transcription factor whose zinc-finger exons occur as distinct isoforms Z1, Z2, Z3, etc.), marks the pupal or metamorphic state. Kamsoi et al. (2021, PNAS 118(34):e2105272118; preprint bioRxiv 2021.03.17.435759) combined morphology, methoprene treatments, and reverse-transcription quantitative PCR in the laboratory-tractable, ovoviviparous (eggs retained within the mother until hatching) mayfly Cloeon dipterum. They found that the Kr-h1 and E93 expression curves intersect at the last nymphal instar, that topical methoprene on freshly ecdysed last-instar nymphs (50 µg) yields Kr-h1 induction, E93 repression, and a supernumerary nymph instead of a subimago (re-forming nymphal mouthparts, central filament — the median caudal appendage of nymphs — and the claw-shaped distal tarsomere, the last segment of the leg), and that forelegs grow dramatically across the last nymphal instar, subimago, and adult. They concluded that mayfly metamorphosis follows the conserved MEKRE93 logic, that metamorphosis is determined before subimago formation (so the subimago is the first of two adult instars), and that distributing necessary adult growth over two instars plausibly explains, at least in part, the adaptive value of the subimago. Crucially, however, the authors' attempts to deplete Kr-h1 and E93 transcripts by RNA interference (RNAi, gene silencing via double-stranded RNA, dsRNA) using multiple dsRNAs failed, so the pathway was validated only by hormone mimicry and expression correlation, not loss of function.

Problem Statement

Resolve the open issues that the Kamsoi et al. (2021) analysis of metamorphosis regulation in the mayfly Cloeon dipterum explicitly left open, whose common objective is a causal developmental-genetic and adaptive account of the mayfly's unique winged molt — the subimago. Specifically: (1) validate, by loss-of-function experiments (RNA interference with demonstrated transcript depletion, or an equivalent causal perturbation), the inferred interaction between Krüppel homolog 1 (Kr-h1) and E93 in Cloeon dipterum — in particular whether E93 depletion produces a supernumerary nymph and Kr-h1 depletion permits precocious metamorphosis, thereby testing whether the MEKRE93 pathway determined from methoprene treatments and expression correlations truly governs the nymph-to-subimago transition and hence whether the subimago is causally an adult instar; (2) complete the inventory of Broad-complex (Br-C) zinc-finger isoforms in C. dipterum, where only Z1, Z2, and Z3 have been identified and additional isoforms are not excluded; (3) determine the relative contribution of the candidate adaptive explanations for retention of the subimago — distribution of necessary adult-structure growth (forelegs and cerci, the paired posterior filaments) across two instars versus hydrofuge (water-repellent) properties of the subimaginal cuticle that ease the water-to-air transition at emergence — including whether they act independently or synergistically; (4) explain the endocrine basis of the mayfly's low juvenile-hormone sensitivity, in which methoprene doses of 0.1-5 µg have no effect on the last-instar nymph and only 50 µg blocks metamorphosis, unlike the dose responses of neopteran insects; and (5) establish through comparative study across Ephemeroptera how labile are the observed trait switches — loss of the nymphal central filament at metamorphosis and the formation of a hook-shaped distal tarsomere at both the subimaginal and the adult molts. Facets (1)-(2) supply the causal genetic specification of the subimago; facets (3)-(5) its adaptive and evolutionary explanation; together they decide whether the 2021 correlation-based account holds as a complete explanation of the subimago stage. These five facets are independently refutable sub-questions: each can be answered and defended on its own. Facet (1) asks for a loss-of-function result with demonstrated transcript depletion, or an equivalent causal perturbation, that tests the Kr-h1-E93 interaction; facet (2) asks whether the Br-C zinc-finger isoform set in C. dipterum is exhaustive — whether Z1-Z3, or a larger set shown to be complete, are the full complement; facet (3) asks for a measured or experimentally decoupled contribution of the two candidate adaptive factors, or a test distinguishing additive from synergistic action; facet (4) asks for the endocrine basis of that low sensitivity and the comparative dose-response evidence that identifies it; and facet (5) asks for comparative data across Ephemeroptera rather than a further C. dipterum-only account.

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