Summary The influence of DNA methylation on gene behavior and its consequent phenotypic effects appear to be very important, but the details are not well understood. Insects offer a diversity of DNA methylation modes, making them an excellent lineage for comparative analyses. However, functional studies have tended to focus on quite specialized holometabolan species, such as wasps, bees, beetles, and flies. Here, we have studied DNA methylation in the hemimetabolan insect Blattella germanica . In this cockroach, a gene involved in DNA methylation, DNA methyltransferase 1 ( DNMT1 ) , is expressed in early embryogenesis. In our experiments, RNAi of DNMT1 reduces DNA methylation and impairs blastoderm formation. Using reduced representation bisulfite sequencing and transcriptome analyses, we observed that methylated genes are associated with metabolism and are highly expressed, whereas unmethylated genes are related to signaling and show low expression. Moreover, methylated genes show greater expression change and less expression variability than unmethylated genes.

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Significance Mayflies are the only extant insects that molt after having formed wings, in a stage called subimago. Numerous authors have wondered whether this stage is a nymph, an adult, or a kind of intermediate. Another question is why mayflies have a subimago stage, when molting a wing is risky. Working with Cloeon dipterum , we found that metamorphosis is regulated as in neopteran insects and that it is determined prior to the formation of the subimago. Thus, it should be considered an instar of the adult stage. We also found that the forelegs grow dramatically between the last nymphal instar, the subimago, and the adult. That necessary growth may help to explain the functional sense of the subimago.

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The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development. Here we show that E93 is also crucial in early embryogenesis in the cockroach, as a significant number of E93-depleted embryos are unable to develop the germ band under maternal RNAi treatment targeting E93 . Moreover, transcriptomic analysis indicates that E93 depletion results in important gene expression changes in the early embryo, and many of the differentially expressed genes are involved in development. Then, using public databases, we gathered E93 expression data in embryo and preadult stages, finding that embryonic expression of E93 is high in hemimetabolan species (whose juveniles, or nymphs, are similar to the adult) and low in holometabolans (whose juveniles, or larvae, are different from the adult). E93 expression is also low in Thysanoptera and in Hemiptera Sternorrhyncha, hemimetabolans with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica , E93 transcript levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. We propose the hypothesis that during evolution, a reduction of E93 expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases of E93 transcripts in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.

The influence of DNA methylation on gene behavior, and its consequent phenotypic effects appear to be very important, but the details are not well understood. Insects offer a diversity of DNA methylation modes, making them an excellent lineage for comparative analyses. However, functional studies have tended to focus on quite specialized holometabolan species, such as wasps, bees, beetles, and flies. Here we have studied DNA methylation in a hemimetabolan insect, the cockroach Blattella germanica, a model of early-branching insects. In this cockroach, one of the main genes responsible for DNA methylation, DNA methyltransferase 1 (DNMT1), is expressed in early embryogenesis. In our experiments, DNMT1 interference by RNAi reduces DNA methylation and impairs blastoderm formation. Using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic analyses, we observed that hypermethylated genes are associated with metabolism and are highly expressed, whereas hypomethylated genes are related to signaling and have low expression levels. Moreover, the expression change in hypermethylated genes is greter than that in hypomethylated genes, whereas hypermethylated genes have less expression variability than hypomethylated genes. The latter observation has also been reported for humans and in Arabidopsis plants. A reduction in expression noise may therefore be one of the few universal effects of DNA methylation.

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In the endopterygote Drosophila melanogaster, Zelda is an activator of the zygotic genome during the maternal‐to‐zygotic transition (MZT). Zelda binds cis‐regulatory elements (TAGteam heptamers), making chromatin accessible for gene transcription. Zelda has been studied in other endopterygotes: Apis mellifera and Tribolium castaneum, and the paraneopteran Rhodnius prolixus. We studied Zelda in the cockroach Blattella germanica, a hemimetabolan, short germ‐band, and polyneopteran species. B. germanica Zelda has the complete set of functional domains, which is typical of species displaying ancestral features concerning embryogenesis. Interestingly, we found D. melanogaster TAGteam heptamers in the B. germanica genome. The canonical one, CAGGTAG, is present at a similar proportion in the genome of these two species and in the genome of other insects, suggesting that the genome admits as many CAGGTAG motifs as its length allows. Zelda‐depleted embryos of B. germanica show defects involving blastoderm formation and abdomen development, and genes contributing to these processes are down‐regulated. We conclude that in B. germanica, Zelda strictly activates the zygotic genome, within the MZT, a role conserved in more derived endopterygote insects. In B. germanica, zelda is expressed during MZT, whereas in D. melanogaster and T. castaneum it is expressed beyond this transition. In these species and A. mellifera, Zelda has functions even in postembryonic development. The expansion of zelda expression beyond the MZT in endopterygotes might be related with the evolutionary innovation of holometabolan metamorphosis. Databases The RNA‐seq datasets of B. germanica, D. melanogaster, and T. castaneum are accessible at the GEO databases https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99785, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18068, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63770, and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84253. In addition, the RNA‐seq library from T. castaneum adult females is available at SRA: SRX021963. The B. germanica reference genome is available as BioProject PRJNA203136.