Establishment of the mayfly Cloeon dipterum as a new model system to investigate insect evolution

Abstract

The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the establishment of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects.

Keywords: Cloeon dipterum; Embryogenesis; Ephemeroptera; Evolutionary and developmental biology; Insect evolution; Mayflies; Paleoptera; Regeneration.

Fig. 1

C. dipterum life cycle. a C. dipterum adult female, b C. dipterum adult male. c Cartoon depicting C. dipterum life cycle. Female lays the eggs in a water stream where they hatch as juvenile nymphs. After several moults nymphs emerge from the water to the land as immature subimagos. Then, they moult again to become sexually mature individuals that fly forming swarms to mate. d Female subimago. e Male subimago. f Early-mid nymph. g Late female nymph. h Late male nymph

Fig. 2

C. dipterum culture in the laboratory. a Couple of adults mating through forced copula. b Temporal sequence of a gravid female laying fertilised eggs that after 94 s hatch as swimming nymphs (white arrowheads). c, d Fertilised eggs and nymphs hatching. e Freshly hatched nymphs. f Culture system in the laboratory. Nymphs are in the beaker with bubbling water and algae. The beaker is placed inside a plastic bottle to keep the subimagos once they emerge from the water. Scale bars: 50 μm

Fig. 3

Representative phases of C. dipterum embryogenesis. Upper panels show embryo morphology detectable through DAPI staining (white). Lower panels show DAPI (nuclei, blue), Actin (cell contour cRed) and mitosis (anti-PH3, green). a–a′ Blastoderm formation (stage 2: st. 2) bcd: blastoderm cells are replicating, shown by PH3 staining (a′), in green. The asterisk highlights a DAPI-rich region located opposite to the embryo with unknown function. b–b′ Germ disc (gd) formation (st. 3). The asterisk highlights a DAPI-rich region located opposite the embryo which we have not identified. It disappears during subsequent stages. c–c′ S-shaped embryo (st. 6). The germ band elongates through active cell proliferation, shown by PH3 staining (abr: abdominal region; hl: head lobe). d–d′ Segmentation of the embryo (st. 8) starts from the cephalic (hl) and thoracic regions, which segments are already visible, towards the abdominal regions (abr). e–e′ Proctodaeum formation (st. 9). Segmentation progresses, appendages enlarge and get segmented (an: antenna, md: mandible, mx: maxilla, lb: labium, pl: pro-leg). f–f′ The abdominal regions are already segmented (abs: abdominal segments). Cercei (ce) are already visible. Dorsal closure proceeds. Scale bars: 50 μm

Fig. 4

C. dipterum embryonic nervous system. a–c Embryo (DAPI staining reveals embryo morphology, b exhibiting the ventral nervous cord (staining using anti-acetylated alpha Tubulin antibody, c). d Surface reconstruction of the ventral nervous cord and its projections towards the appendages. Scale bars: 50 μm

Fig. 5

Genomics and transcriptomic tools. a The genome of C. dipterum is structured in a karyotype of 2n = 10 [95, 96]. Somatic embryonic cell showing condensed DNA in chromosomes (each of them highlighted with an asterisk). DNA stained with DAPI (white) and cell membrane visible through Phalloidin–Rhodamine staining (green). Scale bar: 20 μm. b Pie chart representing top BLASTp results of unigenes against UniRef90 protein database. c otd expression pattern in the mayfly embryo. d en expression domain in C. dipterum embryo

Fig. 6

Leg regeneration of a C. dipterum nymph. a Mayfly nymph before amputation of the third leg. b Nymphal leg (white arrowhead) immediately after amputation. c 24 h after amputation the wound is healed (arrowhead). d 72 h after amputation, the tissue is already partially regenerated. e, f After 7 days, the amputated leg has recovered its initial size and shape with all the segments perfectly formed