Developmental Gene Discovery in a Hemimetabolous Insect: De Novo Assembly and Annotation of a Transcriptome for the Cricket Gryllus Bimaculatus

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 embryos 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.Organismic and Evolutionary Biolog

Comparative Analysis of Cas9 Activators Across Multiple Species

Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression

The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug <it>Oncopeltus fasciatus</it>, 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 <it>O. fasciatus </it>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 <it>de novo </it>transcriptome analyses.</p> <p>Conclusions</p> <p>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.</p> <p>[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (<url>http://www.ncbi.nlm.nih.gov/sra?term=SRP002610</url>). Custom scripts generated are available at <url>http://www.extavourlab.com/protocols/index.html</url>. Seven Additional files are available.]</p

De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis

<p>Abstract</p> <p>Background</p> <p>Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod <it>Daphnia pulex </it>is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod <it>Parhyale hawaiensis</it>. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models.</p> <p>Results</p> <p>To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of <it>P. hawaiensis</it>. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them <it>de novo </it>to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid <it>Acyrthosiphon pisum</it>. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against <b>nr </b>(E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to <it>D. pulex </it>sequences but not to sequences of any other animal. Annotation of several hundred genes revealed <it>P. hawaiensis </it>homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways.</p> <p>Conclusions</p> <p>The amphipod <it>P. hawaiensis </it>has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as <it>D. pulex </it>and <it>P. hawaiensis</it>, as well as the need for development of further substantial crustacean genomic resources.</p

Observations on the breeding biology, nest characteristics, and nest insulation of the Arctic Warbler (Phylloscopus borealis kennicotti) in interior Alaska

In this study, we present the results of two consecutive years of breeding observations of the Arctic Warbler (Phylloscopus borealis kennicotti) in interior Alaska, which represent the most extensive collection of information on this species in North America. The aims of this study were two-fold. First, we described reproductive success across two habitat types. Second, we measured the insulation quality of nests during the second year of our study in order to test whether the substantial variation in nest mass and/or the use of moose hair was related to variation in insulation quality. We observed extremely high rates of nest success in both habitats (collectively 93% of nests fledged at least two chicks). However, we found nest density to be significantly lower and nest failure rate to be significantly higher on thickly vegetated plots that contained higher proportions of dwarf birch shrubs compared to more open plots dominated by willow shrubs. Both nest mass and moose hair mass varied significantly between habitat types, suggesting that Arctic Warblers are able to build nests from a variety of materials, possibly reflecting local availabilities. However, neither nest mass nor moose hair mass was related to insulation quality, reproductive success, or parental quality within either habitat. In addition, insulation quality was not related to any measure of reproductive success, length of nesting period, or parental size. This suggests that widely varying nest types built by Arctic Warblers are capable of providing sufficient insulation to developing chicks

​Supplemental Material for Ewen-Campen and Perrimon, 2018

<div>Figure S1 shows data for control injections into standard nos::Cas9 strains, absent <i>ovoD </i>co-selection. <br></div><div><br></div><div>Table S1 contains sgRNA sequences used in this study.</div><div><br></div><div>Table S2 contains <i>Drosophila</i> genotypes for flies used in this study.</div><div><br></div><div>Table S3 contains experimental details and comprehensive data for each injection.</div

On the origin and evolutionary diversification of beetle horns

Many scarab beetles produce rigid projections from the body called horns. The exaggerated sizes of these structures and the staggering diversity of their forms have impressed biologists for centuries. Recent comparative studies using DNA sequence-based phylogenies have begun to reconstruct the historical patterns of beetle horn evolution. At the same time, developmental genetic experiments have begun to elucidate how beetle horns grow and how horn growth is modulated in response to environmental variables, such as nutrition. We bring together these two perspectives to show that they converge on very similar conclusions regarding beetle evolution. Horns do not appear to be difficult structures to gain or lose, and they can diverge both dramatically and rapidly in form. Although much of this work is still preliminary, we use available information to propose a conceptual developmental model for the major trajectories of beetle horn evolution. We illustrate putative mechanisms underlying the evolutionary origin of horns and the evolution of horn location, shape, allometry, and dimorphism

Statistical comparison of isotig and singleton nucleotide sequence lengths according to BLAST annotation and predicted protein-coding status.

<p>Values shown are <i>p</i>≥0.05 value results of a Welch's t-test.</p>***<p> = <i>p</i><0.0001;</p>*<p><i>p</i><0.05.</p>1<p>BLAST E-value cutoff is e-5 for all hits reported in this table.</p>2<p><b>nr</b> = NCBI non-redundant database.</p>3<p>NRAS = all non-redundant assembly products (isotigs or singletons) regardless of BLAST results against <b>nr</b>.</p>4<p>Numbers of sequences in each category are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061479#pone-0061479-g009" target="_blank">Figure 9</a>. Mean, median, maximum and minimum values for each category are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061479#pone-0061479-t003" target="_blank">Tables 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061479#pone-0061479-t004" target="_blank">4</a>.</p