Mayfly developmental atlas: developmental temporal expression atlas of the mayfly, Ephemera vulgata, reveals short germ-specific hox gene activation.

Background: Over the course of evolution, insects have seen drastic changes in their mode of development. While insects with derived modes of development have been studied extensively, information on ancestral modes of development is lacking. To address this, we selected a member of one of the earliest lineages of extant flying insects, serving as an outgroup to the modern winged insects, the short germ, non-model mayfly Ephemera vulgata Linnaeus (Insecta: Ephemeroptera, Ephemeridae). We document the embryonic morphology throughout its development and establish a global temporal expression atlas.

Results: DAPI staining was used to visualise developmental morphology to provide a frame of reference for the sequenced timepoints. A transcriptome was assembled from 3.2 billion Illumina RNAseq reads divided in 12 timepoints with 3 replicates per timepoint consisting of 35,091 putative genes. We identified 6,091 significantly differentially expressed genes (DEGs) and analysed them for broad expression patterns via gene ontology (GO) as well as for specific genes of interest. This revealed a U-shaped relationship between the sum of DEGs and developmental timepoints, over time, with the lowest number of DEGs at 72 hours after egg laying (hAEL). Based on a principal component analysis of sequenced timepoints, overall development could be divided into four stages, with a transcriptional turning point around katatrepsis. Expression patterns of zld and smg showed a persistent negative correlation and revealed the maternal-to-zygotic transition (MZT), occurring 24 hAEL. The onset of development of some major anatomical structures, including the head, body, respiratory system, limb, muscle, and eye, are reported. Finally, we show that the ancestral short germ sequential mode of segmentation translates to a sequential Hox gene activation and find diverging expression patterns for lab and pb. We incorporate these patterns and morphological observations to an overview of the developmental timeline.

Conclusions: With our comprehensive differential expression study, we demonstrate the versatility of our global temporal expression atlas. It has the capacity to contribute significantly to phylogenetic insights in early-diverging insect developmental biology and can be deployed in both molecular and genomic applications for future research.