Around 150 million years ago, eusocial termites evolved from within the cockroaches, 50 million years before eusocial Hymenoptera, such as bees and ants, appeared. Here, we report the 2-Gb genome of the German cockroach,Blattella germanica, and the 1.3-Gb genome of the drywood termiteCryptotermes secundus. We show evolutionary signatures of termite eusociality by comparing the genomes and transcriptomes of three termites and the cockroach against the background of 16 other eusocial and non-eusocial insects. Dramatic adaptive changes in genes underlying the production and perception of pheromones confirm the importance of chemical communication in the termites. These are accompanied by major changes in gene regulation and the molecular evolution of caste determination. Many of these results parallel molecular mechanisms of eusocial evolution in Hymenoptera. However, the specific solutions are remarkably different, thus revealing a striking case of convergence in one of the major evolutionary transitions in biological complexity.

Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricketGryllus bimaculatus, and the annotation of the 1.60-Gb genome of the Hawaiian cricketLaupala kohalensis. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of thepickpocketclass V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping.

Is there a correlation between miRNA diversity and levels of organismic complexity? Exhibiting extraordinary levels of morphological and developmental complexity, insects are the most diverse animal class on earth. Their evolutionary success was in particular shaped by the innovation of holometabolan metamorphosis in endopterygotes. Previously, miRNA evolution had been linked to morphological complexity, but astonishing variation in the currently available miRNA complements of insects made this link unclear. To address this issue, we sequenced the miRNA complement of the hemimetabolan Blattella germanica and reannotated that of two other hemimetabolan species, Locusta migratoria and Acyrthosiphon pisum, and of four holometabolan species, Apis mellifera, Tribolium castaneum, Bombyx mori and Drosophila melanogaster. Our analyses show that the variation of insect miRNAs is an artefact mainly resulting from poor sampling and inaccurate miRNA annotation, and that insects share a conserved microRNA toolkit of 65 families exhibiting very low variation. For example, the evolutionary shift toward a complete metamorphosis was accompanied only by the acquisition of three and the loss of one miRNA families.

The success of neopteran insects, with 1 million species described, is associated with developmental innovations such as holometaboly and the evolution from short to long germband embryogenesis. To unveil the mechanisms underlining these innovations, we compared gene expression during the ontogeny of two extreme neopterans, the cockroach Blattella germanica (polyneopteran, hemimetabolan, and short germband species) and the fly Drosophila melanogaster (endopterygote, holometabolan, and long germband species). Results revealed that genes associated with metamorphosis are predominantly expressed in late nymphal stages in B. germanica and in the early-mid embryo in D. melanogaster. In B. germanica the maternal to zygotic transition (MZT) concentrates early in embryogenesis, when juvenile hormone factors are significantly expressed. In D. melanogaster, the MZT extends throughout embryogenesis, during which time juvenile hormone factors appear to be unimportant. These differences possibly reflect broad trends in the evolution of development within neopterans, related to the germband type and the metamorphosis mode.

Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0–6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.

The early embryo of the cockroachBlattella germanicaexhibits highE93expression. 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 targetingE93. 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 gatheredE93expression data in embryo and preadult stages, finding that embryonic expression ofE93is 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).E93expression 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 ZygentomaThermobia domestica,E93transcript 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 ofE93expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases ofE93transcripts 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.