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It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction.

The Nusplingen Plattenkalk is a Solnhofen-type fossil Konservat-Lagerstaette in the southwestern part of the Swabian Alb, which is scientifically exploited by the Stuttgart Natural History Museum. The Nusplingen Plattenkalk formed in a deep lagoon surrounded by islands. The highly diverse and exceptionally preserved fossil fauna and flora allow a reconstruction of the Late Jurassic marine food-web and the palaeoenvironment. The complete outcrop area is preserved as a national heritage site within the Swabian Alb Geopark. Information for the public is provided by guided tours, a geological trail with a small public sampling site and fossil exhibitions.

Background Most evolutionary developmental biology (\"evo-devo\") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery. Results We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus , which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses. Conclusions Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms. [The sequence data from this study have been submitted to GenBank under study accession number SRP002610 ( http://www.ncbi.nlm.nih.gov/sra?term=SRP002610 ). Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html . Seven Additional files are available.]

Background Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative—to some degree—for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. Results Here, we systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (~ 10%) or beetle (~ 40%) reflecting both technical and biological differences. Accordingly, we were able to annotate novel developmental GO terms for 96 genes studied in this work. With this work, we publish the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). Conclusions We conclude that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor.

Dorsoventral pattering relies on Toll and BMP signalling in all insects studied so far, with variations in the relative contributions of both pathways. Drosophila and the beetle Tribolium share extensive dependence on Toll, while representatives of more distantly related lineages like the wasp Nasonia and bug Oncopeltus rely more strongly on BMP signalling. Here, we show that in the cricket Gryllus bimaculatus , an evolutionarily distant outgroup, Toll has, like in Drosophila , a direct patterning role for the ventral half of the embryo. In addition, Toll polarises BMP signalling, although this does not involve the conserved BMP inhibitor Sog/Chordin. Finally, Toll activation relies on ovarian patterning mechanisms with striking similarity to Drosophila . Our data suggest two surprising hypotheses: (1) that Toll's patterning function in Gryllus and Drosophila is the result of convergent evolution or (2) a Drosophila-like system arose early in insect evolution and was extensively altered in multiple independent lineages.

The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, we used comparative genomics, parental RNAi, and gene expression analyses in multiple insect species. We have found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. Our results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. We propose that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program.

Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos. In spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders. Despite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression. To address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate. At the earliest stages of animal development, embryos consisting of only a handful of cells must figure out where each part of their body will come from. The first step in this process is to determine what will be their head versus their tail, and what will be their front versus their back. Many animals use specialized groups of cells, called “organizers”, to make this decision. This occurs in backboned animals – including humans – and also in distantly related animals such as spiders. In spiders, the developing embryo must form an organizer called the “cumulus” or the spiderling will not develop correctly. In order to form and maintain the cumulus, various genes must be turned on in a carefully controlled order in exactly the right cells. Pechmann et al. have now discovered the role of a previously unknown gene (called Pt-Ets4) that marks the spot where the cumulus forms. This gene is required for cumulus maintenance and it also helps to activate a number of other cumulus-specific genes. When this gene is disrupted, the spider embryo does not properly differentiate its front from its back. The findings presented by Pechmann et al. add to a growing foundation of studies aiming to understand how genes ‘talk’ to one another and organize embryos as they develop. In years to come, the unraveling of these gene pathways, where genes sequentially turn other genes on and off, will allow us to more fully understand how a single cell can grow into a complete adult animal.

The desert locust (Schistocerca gregaria) represents one of the most destructive agricultural pests globally, renowned for its ability to form massive swarms that can devastate crops and threaten food security across vast regions. Despite the widespread application of the CRISPR/Cas9 gene-editing system in several insect orders, its utilization in locusts, particularly in the desert locust, has remained relatively unexplored. We established a CRISPR/Cas9-mediated gene-editing workflow for the desert locust using gene encoding for neuropeptide corazonin (Crz) as a target. We also analyzed the phenotypic and physiological characteristics of the mutant using paraffin sectioning, HE staining, and chitin staining techniques. Our findings revealed that while Crz knockout desert locusts were viable and maintained normal fertility, they exhibited striking phenotypic alterations, including albinism and a significant reduction in cuticle thickness. These observations not only highlight the functional role of Crz in pigmentation and cuticle development but also underscore the potential of CRISPR/Cas9 as a powerful tool for dissecting gene function in locusts. Furthermore, the successful application of CRISPR/Cas9 in desert locusts also paves the way for similar genetic studies in other non-model insects, expanding the scope of functional genomics in entomology.

The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, here we show in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning, and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fog’s role during cellularization is widely conserved and therefore might represent the ancestral function of the pathway.

Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.