Developmental transcriptomics of the Firebrat: Exploring developmental expression patterns and morphology during the embryogenesis of Thermobia domestica

Abstract

Understanding the development of early-diverging lineages is crucial for inferring evolutionary context in evolutionary developmental biology. Thermobia domestica (the firebrat), a member of Zygentoma, holds particular significance to insect phylogenetics due to its position as a sister group to all winged insects (Pterygota). We explore its development by reporting on the embryonic morphology using DAPI staining at 14 selected timepoints throughout development, which lasts 10 days. At each timepoint, RNA sequencing was conducted to perform a large-scale transcriptomic analysis. Differential gene expression analysis and gene ontology (GO) enrichment studies revealed global expression patterns and linked biological processes to specific developmental stages. Key findings include the identification of major transcriptional turning points during three developmental stages: The maternal to zygotic transition between 16 and 24 hours after egg-laying (hAEL), katatrepsis between 72 and 96 hAEL, and hatching between 216 and 240 hAEL. Additionally, the GO study mapped the timing of biological processes, such as cleavage, blastoderm formation, dorsal closure, and organogenesis, particularly tracheal and muscular development. These insights establish a robust temporal framework for T. domestica embryogenesis and provide a foundation for inferring ancestral and derived developmental traits in insects. The dataset serves as a valuable library for genetic studies and reference for comparative evo-devo studies with other insect species.

Fig 1. The embryogenesis of Thermobia domestica visualised with DAPI staining.

(A-M) Lateral views of DAPI-stained embryos at successive sampled timepoints in hours after egg-laying (hAEL). (N) Brightfield image in colour of a hatched T. domestica nymph. Scale bar (bottom right) = 100 μm. ant, antenna; cc, cercus; cf, caudal filament; gd, germ disc; l1-3, prothoracic, mesothoracic, and metathoracic legs, respectively; lb, labium; md, mandible; mx, maxilla; pce, protocephalon; pco, protocorm; ser, serosa. Numbers indicate abdominal segments.

Fig 2. Differential expression of Thermobia domestica development.

(A) Principal components 1 and 2 of a principal component analysis (PCA) plot depicting sample relatedness of biological replicates within sequenced timepoints in hours after egg-laying (hAEL). Arrows depict turning points in gene expression. (B) A heatmap displaying TMM- and log2-normalised, scaled gene expression values, hierarchically clustered based on gene expression pattern similarity. Dendrogram and cluster numbers are shown on the left and right sides of the heatmap, respectively.

Fig 3. Expression patterns of transcripts representing the maternal to zygotic transition (MZT).

The expression levels of transcripts from clusters 37, 40, 42, 43, 44, and 45, annotated as maternally deposited throughout development in hours after egg-laying (hAEL). These are selected based on their association with the MZT and the expression of smaug (smg), associated with maternal transcript depletion, is highlighted. Mean expression values are indicated in black. Expression data is presented in log2-centred TMM (+1) values.

Fig 4. Expression patterns and GOslim enrichments associated with clusters 36, 37, 40, 42, 43 and 45.

Selected GOslim enrichments are displayed based on relevance to the described processes. Terms above the horizontal line are described in the main text. Stars (*) indicate that the cluster is uniquely enriched for the concerning term. Expression data throughout development in hours after egg-laying (hAEL) is presented in log2-centred TMM (+1) values. Mean expression values are indicated in black.

Fig 5. Expression patterns and GOslim enrichments associated with clusters 4, 6, 8, 19, 33 and 34.

Selected GOslim enrichments are displayed based on relevance to the described processes. Terms above the horizontal line are described in the main text. Stars (*) indicate that the cluster is uniquely enriched for the concerning term. Expression data throughout development in hours after egg-laying (hAEL) is presented in log2-cent-red TMM (+1) values. Mean expression values are indicated in black.