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Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria . For Oncopeltus , RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays and describe two novel AMP families called Serosins and Ovicins. For both insects, quantitative polymerase chain reaction shows immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects. This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

Insects have been extraordinary successful in colonizing terrestrial habitats and this success is partly due to a protective cuticle that mainly contains chitin and proteins. The cuticle has been well studied in larvae and adults, but little attention has been paid to the cuticle of the egg. This cuticle is secreted by the serosa, an extraembryonic epithelium that surrounds the yolk and embryo in all insect eggs, but was lost in the Schizophoran flies to which Drosophila belongs. We therefore set out to investigate serosal cuticle formation and function in a beetle (Tribolium castaneum) using RNAi-mediated knockdown of three candidate genes known to structure chitin in the adult cuticle, and we aimed to identify other serosal cuticle genes using RNA sequencing. Knockdown of Knickkopf (TcKnk-1) or Retroactive (TcRtv) affects the laminar structure of the serosal cuticle, as revealed by Transmission Electron Microscopy in knockdown eggs. In the absence of this laminar structure, significantly fewer eggs survive at low humidity compared to wild-type eggs. Survival in dry conditions is also adversely affected when cross-linking among proteins and chitin is prevented by Laccase2 (TcLac-2) RNAi. Finally, we compare the transcriptomes of wild-type eggs to serosa-less eggs and find serosa-biased expression of 21 cuticle-related genes including structural components, chitin deacetylases and chitinases. Our data indicate that the serosal cuticle utilizes the same machinery for structuring the cuticle as adults. We demonstrate that the structure of the cuticle is crucial for desiccation resistance, and we put forward the serosal cuticle of Tribolium as an excellent model to study the ecological properties of the insect cuticle.

Innate immunity is common to all metazoans and serves as a first line of defense against pathogens. Although the immune response of adult and larval insects has been well characterized, it remains unknown whether the insect egg is able to mount an immune response. Contrary to Drosophila, Tribolium eggs develop an extraembryonic epithelium, the serosa. Epithelia are well known for their ability to fight infection, so the serosa has the potential to protect the embryo against pathogens. To test this hypothesis we created serosa-less eggs by Tc-zen1 parental RNAi. We found that the Tribolium egg upregulates several immune genes to comparable levels as adults in response to infection. Drosophila eggs and serosa-less Tribolium eggs, however, show little to no upregulation of any of the tested immune genes. We conclude that the extraembryonic serosa is crucial for the early immune competence of the Tribolium egg.

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.

In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophilamelanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Triboliuminnexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.

Drosophila larvae and adults possess a potent innate immune response, but the response of Drosophila eggs is poor. In contrast to Drosophila, eggs of the beetle Tribolium are protected by a serosa, an extraembryonic epithelium that is present in all insects except higher flies. In this study, we test a possible immune function of this frontier epithelium using Tc-zen1 RNAi-mediated deletion. First, we show that bacteria propagate twice as fast in serosa-less eggs. Then, we compare the complete transcriptomes of wild-type, control RNAi, and Tc-zen1 RNAi eggs before and after sterile or septic injury. Infection induces genes involved in Toll and IMD-signaling, melanisation, production of reactive oxygen species and antimicrobial peptides in wild-type eggs but not in serosa-less eggs. Finally, we demonstrate constitutive and induced immune gene expression in the serosal epithelium using in situ hybridization. We conclude that the serosa provides insect eggs with a full-range innate immune response. DOI: http://dx.doi.org/10.7554/eLife.04111.001, eLife digest Insects are among the most numerous and diverse creatures on Earth, and over a million different species of insects have been described. Insects have a hard exoskeleton that protects their segmented bodies, and adult insects and their young are also well protected from pathogens. To fight off infection by bacteria or viruses, these creatures release antimicrobial molecules in the fluid that bathes their internal organs. Insects can also mount a localized immune response that kills off invading microbes. Most of what scientists have learned about the insect immune system has come from studying fruit flies. While much of the knowledge gained has been applicable to other insects, there is an important exception—fruit fly eggs are incredibly vulnerable to infection. Eggs from other insects are far better protected. In some species, the mother insect protects her eggs either through scrupulous care or by coating them with her own antimicrobial fluids. However, it was unclear if insect eggs could also defend themselves and counter an infection with a strong immune response. To better understand the immune response in insect eggs, Jacobs et al. studied the eggs of red flour beetles. These beetles are common agricultural pests that eat stored grains and are often studied by scientists in the laboratory. The beetle eggs share a trait with all other insect eggs that is missing from fruit flies and some other flies; the beetle eggs have an extra layer—called the serosa—that envelops the yolk and the developing embryo. To test whether this extra layer provides immune protection for the egg, Jacobs et al. used a technique called RNA interference to prevent the formation of the serosa. Beetle eggs either with or without a serosa were then pricked with a bacteria-covered object, and Jacobs et al. observed that the bacteria grew twice as fast in the eggs lacking a serosa compared with the eggs that had a serosa. Next, Jacobs et al. examined gene expression in response to the infection in the eggs. Over 500 genes that are expressed after an infection were identified, and of these genes, 481 were only expressed in eggs with a serosa. Three of these genes, including two that encode antimicrobial molecules, were looked at in more detail, and found to be only expressed within the serosa, indicating that the serosa is the most likely source of the egg's immune response. Importantly, Jacobs et al. found that eggs with a serosa produce the same immune system response as adult insects and concluded that most insect eggs are far from defenseless and are capable of fending off infection. DOI: http://dx.doi.org/10.7554/eLife.04111.002

The cytokines of the TGFbeta superfamily are highly conserved in evolution and elicit a diverse range of cellular responses in all metazoa. In Drosophila, the signaling pathways of the two TGFbeta subfamilies, Activins and Bone Morphogenetic Proteins (BMPs), have been well studied. To address the question of whether the findings from Drosophila are representative of insects in general, we analyzed the components of TGFbeta-signaling present in the genome of the beetle Tribolium castaneum. We were able to identify orthologs of the BMPs Decapentaplegic and Glass bottom boat, of the Activins Activinbeta and Dawdle, as well as orthologs of the less well-known ligands Myoglianin and Maverick, together with orthologs of all TGFbeta receptors and cytoplasmic signal transducers present in Drosophila. This indicates that the diversity of TGFbeta signaling components is generally well conserved between Drosophila and Tribolium. However, the genome of the beetle-and of the bee Apis mellifera-lacks an ortholog of the Drosophila BMP Screw but does contain a vertebrate-like BMP10 homolog which is not found in Drosophila. Concerning BMP inhibitors, Tribolium displays an even more vertebrate-like ensemble of components. We found two orthologs of the vertebrate DAN family, Dan and Gremlin, and show embryonic expression of a vertebrate-like BAMBI ortholog, all of which are absent in Drosophila. This suggests that Tribolium might have retained a more ancestral composition of TGFbeta signaling components and that TGFbeta signaling underwent considerable change in the Drosophila lineage. Tribolium is an excellent model to study the function of these ancestral signaling components in insects.

Bone morphogenetic protein (BMP) signaling plays a major role in dorsoventral patterning in vertebrates and in Drosophila . Remarkably, in Tribolium , a beetle with an ancestral type of insect development, early BMP/ dpp exhibits differential expression along the anteroposterior axis. However, the BMP/Dpp inhibitor Sog/chordin is expressed ventrally and establishes a dorsal domain of BMP/Dpp activity by transporting BMPs toward the dorsal side, like in Drosophila . Loss of Tribolium Sog not only abolishes dorsoventral polarity in the ectoderm, but also leads to the complete absence of the CNS. This phenotype suggests that sog is the main BMP antagonist in Tribolium , in contrast to vertebrates and Drosophila , which possess redundant antagonists. Surprisingly, Sog also is required for head formation in Tribolium , as are the BMP antagonists in vertebrates. Thus, in Tribolium , the system of BMP and its antagonists is less complex than in Drosophila or vertebrates and combines features from both, suggesting that it might represent an ancestral state.

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane byzerknüllt1RNAi in the beetleTribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression ofTc-chitin-synthase1and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates.

Summary Background: In the long-germ insect Drosophila , a single extraembryonic membrane, the amnioserosa, covers the embryo at the dorsal side. In ancestral short-germ insects, an inner membrane, the amnion, covers the embryo ventrally, and an outer membrane, the serosa, completely surrounds the embryo. An early differentiation step partitions the uniform blastoderm into the anterior-dorsal serosa and the posterior-ventral germ rudiment giving rise to amnion and embryo proper. In Drosophila , amnioserosa formation depends on the dorsoventral patterning gene zerknu¨llt ( zen ), a derived Hox3 gene. Results: The short-germ beetle Tribolium castaneum possesses two zen homologs, Tc-zen1 and Tc-zen2 . Tc-zen1 acts early and specifies the serosa. The loss of the serosa after Tc-zen1 RNAi is compensated by an expansion of the entire germ rudiment toward the anterior. Instead of the serosa, the amnion covers the embryo at the dorsal side, and later size regulation normalizes the early fate shifts, revealing a high degree of plasticity of short-germ development. Tc-zen2 acts later and initiates the amnion and serosa fusion required for dorsal closure. After Tc-zen2 RNAi, the amnion and serosa stay apart, and the embryo closes ventrally, assuming a completely everted (inside-out) topology. Conclusions: In Tribolium , the duplication of the zen genes was accompanied by subfunctionalization. One of the paralogues, Tc-zen1 , acts as an early anterior-posterior patterning gene by specifying the serosa. In absence of the serosa, Tribolium embryogenesis acquires features of long-germ development with a single extraembryonic membrane. We discuss implications for the evolution of insect development including the origin of the zen -derived anterior determinant bicoid .