Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome.

BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes.

The beetle amnion and serosa functionally interact as apposed epithelia.

Unlike passive rupture of the human chorioamnion at birth, the insect extraembryonic (EE) tissues - the amnion and serosa - actively rupture and withdraw in late embryogenesis. Withdrawal is essential for development and has been a morphogenetic puzzle. Here, we use new fluorescent transgenic lines in the beetle Tribolium castaneum to show that the EE tissues dynamically form a basal-basal epithelial bilayer, contradicting the previous hypothesis of EE intercalation. We find that the EE tissues repeatedly detach and reattach throughout development and have distinct roles. Quantitative live imaging analyses show that the amnion initiates EE rupture in a specialized anterior-ventral cap. RNAi phenotypes demonstrate that the serosa contracts autonomously. Thus, apposition in a bilayer enables the amnion as 'initiator' to coordinate with the serosa as 'driver' to achieve withdrawal. This EE strategy may reflect evolutionary changes within the holometabolous insects and serves as a model to study interactions between developing epithelia.

Visualizing late insect embryogenesis: extraembryonic and mesodermal enhancer trap expression in the beetle Tribolium castaneum.

The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue's rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.

Regulation of expression of Vg and establishment of the dorsoventral compartment boundary in the wing imaginal disc by Suppressor of Hairless.

The transcription factor Suppressor of Hairless (Su(H)) belongs to the CSL transcription factor family, which are the main transcriptional effectors of the Notch-signaling pathway. Su(H) is the only family member in the Drosophila genome and should therefore be the main transcriptional effector of the Notch pathway in this species. Despite this fact, in many developmental situations, the phenotype caused by loss of function of Su(H) is too weak for a factor that is supposed to mediate most or all aspects of Notch signaling. One example is the Su(H) mutant phenotype during the development of the wing, which is weaker in comparison to other genes required for Notch signaling. Another example is the complete absence of a phenotype upon loss of Su(H) function during the formation of the dorsoventral (D/V) compartment boundary, although the Notch pathway is required for this process. Recent work has shown that Su(H)/CBF1 has a second function as a transcriptional repressor, in the absence of the activity of the Notch pathway. As a repressor, Su(H) acts in a complex together with Hairless (H), which acts as a bridge to recruit the co-repressors Groucho and CtBP, and acts in a Notch-independent manner to prevent the transcription of target genes. This raises the possibility that a de-repression of target genes can occur in the case of loss if function of Su(H). Here, we show that the weak phenotype of Su(H) mutants during wing development and the absence of a phenotype during formation of the D/V compartment boundary are caused by the concomitant loss of the Notch-independent repressor function. This loss of the repressor function of Su(H) results in a de-repression of expression of target genes to a different degree in each process. Loss of Su(H) function during wing development results in a transient de-repression of expression of the selector gene vestigial (vg). We show that this residual expression of vg is responsible for the weaker mutant phenotype of Su(H) in the wing. During the formation of the D/V compartment boundary, de-repression of target genes seems to be sufficiently strong, to compensate the loss of Su(H) activity. Thus, de-repression of its target genes obscures the involvement of Su(H) in this process. Furthermore, we provide evidence that Dx does not signal in a Su(H)-independent manner as has been suggested previously.

A Notch-independent function of Suppressor of Hairless during the development of the bristle sensory organ precursor cell of Drosophila.

Su(H)/CBF1 is a key component of the evolutionary conserved Notch signalling pathway. It is a transcription factor that acts as a repressor in the absence of the Notch signal. If Notch signalling is activated, it associates with the released intracellular domain of the Notch receptor and acts as an activator of transcription. During the development of the mechanosensory bristles of Drosophila, a selection process called lateral inhibition assures that only a few cells are selected out of a group to become sensory organ precursors (SOP). During this process, the SOP cell is thought to suppress the same fate in its surrounding neighbours via the activation of the Notch/Su(H) pathway in these cells. We show that, although Su(H) is required to prevent the SOP fate during lateral inhibition, it is also required to promote the further development of the SOP once it is selected. Importantly, in this situation Su(H) appears to act independently of the Notch signalling pathway. We find that loss of Su(H) function leads to an arrest of SOP development because of the loss of sens expression in the SOP. Our results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. We show that this repressor activity is encoded by one or several genes of the E(spl)-complex. Our results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl.