Immediate-Early Promoter-Driven Transgenic Reporter System for Neuroethological Research in a Hemimetabolous Insect.
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ABSTRACT:
SUBMITTER: Watanabe T
PROVIDER: S-EPMC6140108 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mechanism of sex determination remains poorly understood in hemimetabolous insects. Here, in the brown planthopper (BPH), Nilaparvata lugens, a hemipteran rice pest, we identified a feminizing switch or a female determiner (Nlfmd) that encodes a serine/arginine-rich protein. Knockdown of Nlfmd in female nymphs resulted in masculinization of both the somatic morphology and doublesex splicing. The female-specific isoform of Nlfmd, Nlfmd-F, is maternally deposited and zygotically transcribed. Depletion of Nlfmd by maternal RNAi or CRISPR-Cas9 resulted in female-specific embryonic lethality. Knockdown of an hnRNP40 family gene named female determiner 2 (Nlfmd2) also conferred masculinization. In vitro experiments showed that an Nlfmd2 isoform, NlFMD2340, bound the RAAGAA repeat motif in the Nldsx pre-mRNA and formed a protein complex with NlFMD-F to modulate Nldsx splicing, suggesting that NlFMD2 may function as an RNA binding partner of the feminizing switch NlFMD. Our results provide novel insights into the diverse mechanisms of insect sex determination.
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Project description:The unparalleled success of the insects comprising more than a million species has long stood out to evolutionary biologists. A much overlooked evolutionary innovation of the insects is the serosa, an extraembryonic epithelium that covers yolk and embryo in their eggs. We have shown that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, if this innate immune competence evolved in the Tribolium lineage, or is ancestral to all insects. Here, we expand our studies to the bug Oncopeltus fasciatus that belongs to the basal main group of insects, the Hemimetabola. RNA sequencing reveals an extensive transcriptional response upon infection of the egg with Gram-positive and Gram-negative bacteria. We demonstrate the antimicrobial activity of upregulated peptides using in vitro bacterial growth inhibition assays, and describe two novel families of AMPs called Serosins and Ovicins. By qPCR, we determine that eggs become immune responsive when the serosa develops. Finally, in situ hybridizations show that transcripts of upregulated peptides are located in the serosal cells and not in the underlying embryo. We conclude that the serosa protects the O. fasciatus embryo against pathogens. 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 been one of the factors that facilitated the spectacular success of the insects.
2019-01-01 | GSE100429 | GEO
Project description: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'.
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Project description:In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3' end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex.
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Project description:Insects display a whole spectrum of morphological diversity, which is especially noticeable in the organization of their appendages. A recent study in a hemipteran, Oncopeltus fasciatus (milkweed bug), showed that nubbin (nub) affects antenna morphogenesis, labial patterning, the length of the femoral segment in legs, and the formation of a limbless abdomen. To further determine the role of this gene in the evolution of insect morphology, we analyzed its functions in two additional hemimetabolous species, Acheta domesticus (house cricket) and Periplaneta americana (cockroach), and re-examined its role in Drosophila melanogaster (fruit fly). While both Acheta and Periplaneta nub-RNAi first nymphs develop crooked antennae, no visible changes are observed in the morphologies of their mouthparts and abdomen. Instead, the main effect is seen in legs. The joint between the tibia and first tarsomere (Ta-1) is lost in Acheta, which in turn, causes a fusion of these two segments and creates a chimeric nub-RNAi tibia-tarsus that retains a tibial identity in its proximal half and acquires a Ta-1 identity in its distal half. Similarly, our re-analysis of nub function in Drosophila reveals that legs lack all true joints and the fly tibia also exhibits a fused tibia and tarsus. Finally, we observe a similar phenotype in Periplaneta except that it encompasses different joints (coxa-trochanter and femur-tibia), and in this species we also show that nub expression in the legs is regulated by Notch signaling, as had previously been reported in flies and spiders. Overall, we propose that nub acts downstream of Notch on the distal part of insect leg segments to promote their development and growth, which in turn is required for joint formation. Our data represent the first functional evidence defining a role for nub in leg segmentation and highlight the varying degrees of its involvement in this process across insects.
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