Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera)

Abstract

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.

Keywords: insect development; insect endocrinology; insect evolution; insect metamorphosis.

Fig. 1.

Last nymphal instars and wing maturation in Cloeon dipterum. (A) Female nymph in the preantepenultimate instar (PAN); the wing pads do not reach the first abdominal segment (A1). (B) Female nymph in the antepenultimate instar (AN); the wing pads just reach the anterior edge of A1. (C) Male nymph in the penultimate instar (PN); the wing pads go beyond the anterior edge of A1. (D) Female nymph in the last instar (LN); the wing pads go beyond A2. (E–G) Development of the wing pads in the LN; shape and color in 3-d-old nymphs (LND3) (E), early 4 d old (LND4e) (F), and late 4 d old (LND4l) (G). (H) Wing primordia in the PN. (I) Wing in LN4l, folded within the wing pad. (J) Wing in LND4l, removed from the wing pad and extended on a slide. (K) Apical part of a subimago wing. (L) Apical part of an adult wing. From A to D, images obtained on the first day of the corresponding instar; the perimeter of the wing pads has been indicated with a solid line, and the anterior edge of A1 with a blue line. (Scale bars: 1 mm.)

Fig. 2.

The mouthparts, central filament, and legs during Cloeon dipterum metamorphosis. (A) The head of a female in the last day of the penultimate nymphal instar (PN) showing the corresponding mouthparts, and a new set of mouthparts (exemplified by a new pair of mandibles and a maxila) that are being formed, which corresponds to the last nymphal instar (LN). (B) Head of a male subimago showing that the mouthparts disappeared (circle). (C) PN with two cerci and a central filament. (D) Detail of the cerci and filament in PN. (E) Late LN (black wing pads stage) showing the two cerci and the central filament. (F) Detail of the cerci and filament in a late LN after the apolysis; note the new cerci formed (yellow arrowhead), whereas the central filament only shows the nymphal structure (red arrowhead). (G) Habitus of a female subimago. (H) Detail of the distal tarsomere in 4-d-old LN (LND4); note the actual claw and the small hook-shaped structure being formed after the apolysis, which corresponds to the subimago (arrowhead). (I) Detail of the distal tarsomere in the adult. (J) Foreleg of a male LN, subimago, and adult. (Scale bars: 0.25 mm in H and I; 0.5 mm in A, B, D, and J; 1 mm in C, E, F, and G.)

Fig. 3.

Gene expression in Cloeon dipterum. (A) Genes relevant for metamorphosis; the genes Met, Kr-h1, E93, HR3, and Br-C were examined; in the latter gene, we measured the expression of each of the isoforms, Br-C Z1, Br-C Z2, and Br-CZ3, and the joint expression of all the isoforms (Br-C core); the expression was measured in females of preantepenultimate nymphal instar (PAN), antepenultimate nymphal instar (AN), penultimate nymphal instar (PN), all days of the last nymphal instar (LN, from D0 to D4), the subimago (SI), and the adult (Ad). (B) Expression of Vg in the same stages. (C) Expression of Vg in the female (F) and male (M) subimago. (D) Effect of methoprene on Vg expression; freshly ecdysed last instar female nymphs were treated with methoprene (T) or with acetone (C), and Vg expression was measured 2 d later. The results are indicated as copies of the examined mRNA per 1,000 copies of CdActin-5c mRNA and are expressed as the mean ± SEM (n = 3 biological replicates). The asterisks in C and D indicate statistically significant differences between samples (P < 0.01) according to the REST (33).

Fig. 4.

Effects of methoprene on metamorphosis in Cloeon dipterum. Methoprene was administered in freshly ecdysed last instar nymphs (LND0), and morphological features were examined 4 d later (LND4) in controls and methoprene-treated insects. (A) Mouthparts; note that the controls only show the nymphal structures, whereas the methoprene-treated show the nymphal structures and a new set formed after the apolysis; H: lateral lobe of the hypopharynx, Li: labial complex; Lr: labrum; Mn: mandible. (B) Cerci and central filament; note that the controls form new cerci but they do not form the central filament (black arrowhead), whereas the methoprene-treated insects form the cerci and the central filament (red arrowhead). (C) Distal tarsomere exemplified by the hind leg; note that the controls form the new structure, short and hook-shaped, which is characteristic of the subimago (black arrowhead), whereas the methoprene-treated insects form again the elongated and claw-shaped structure that is characteristic of the nymphal instars (red arrowhead). (D) Developing wings; the general morphology and vein pattern are similar in controls and in methoprene-treated. (Scale bars: 0.5 mm in A and C, 1 mm in B, and 2 mm in D.)

Fig. 5.

Effects of methoprene treatment on the expression of genes involved in metamorphosis in Cloeon dipterum. Methoprene was administered in freshly ecdysed last instar female nymphs, and gene expression was measured in the head, wing pads, abdominal epidermis, and ovaries 2 d later. The genes Kr-h1, E93, and Br-C were examined; in the latter gene, we measured the expression of each of the isoforms, Br-C Z1, Br-C Z2, and Br-C Z3, and the joint expression of all the isoforms (Br-C core). The results are indicated as copies of the examined mRNA per 1,000 copies of CdActin-5c mRNA and are expressed as the mean ± SEM (n = 3 biological replicates); the asterisk indicates statistically significant differences between methoprene-treated and control insects (P < 0.05) according to the REST (33).