dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgments All authors acknowledge the hospitality of the  Lorentz Center  for international workshops (Leiden, The Netherlands), where the idea of this work was born, and acknowledge the support of the  International Space Science Institute  ( ISSI Bern, Switzerland ), where the collaboration was originated. We thank the reviewers for having provided valuable and constructive comments that improved the clarity of the manuscript, and  J. E. Barnes ,  L. Blecha ,  M. Eracleous ,  B. D. Farris ,  H. Fu ,  X. Liu ,  R. Pfeifle ,  L. C. Popovic ,  C. Ricci ,  S. Rodriguez ,  S. Tang ,  G. B. Taylor  for their kind permission to reuse figures from their publications. ADR, CV and SB acknowledge financial support from  ASI  under grant  ASI-INAF I/037/12/0 , and from the agreement ASI-INAF n.
2017-14-H. O. TB acknowledges support by the  National Aeronautics and Space Administration  under Grant No. NNX15AK84G  and  80NSSC19K0319  issued through the  Astrophysics Theory Program  and by the  Research Corporation for Science Advancement  through a  Cottrell Scholar Award. SF and KÉG thank the  Hungarian National Research, Development, and Innovation Office  ( OTKA NN110333 ) for support. KÉG was supported by the  János Bolyai Research Scholarship of the Hungarian Academy of Sciences  and by the ÚNKP-19-4  New National Excellence Program  of the  Ministry for Innovation and Technology. BH  acknowledges financial support by the  DFG  grant  GE625/17-1. EL is supported by a  European Union   CO-FUND/Durham Junior Research Fellowship (under EU  grant agreement no. 609412 ). DL acknowledges support from the  European Research Council (ERC)  under grant  647208  (PI Jonker).
KI acknowledges support by the  Spanish MINECO  under grant  AYA2016-76012-C3-1-P  and  MDM-2014-0369  of  ICCUB (Unidad de Excelencia 'María de Maeztu ' ). MPT acknowledges financial support from the  Spanish MCIU through the "Center of Excellence Severo Ochoa " award for the  Instituto de Astrofísica de Andalucía  ( SEV-2017-0709 ) and through the  MINECO grants   AYA2012-38491-C02-02  and  AYA2015-63939-C2-1-P.
The data were normalized to protein content measured in each well using BCA assay (Thermo Fisher Scientific) according to the manufacturer's instructions. Seahorse parameters were calculated as followed: last rate measurement before oligomycin injection -minimum rate measurement after oligomycin injection = ATP production; last rate measurement before first injection -non-mitochondrial respiration rate = basal respiration; maximum rate measurement after FCCP injection -non-mitochondrial respiration = maximal respiration. To determine the contribution of fatty acid oxidation to the OCR, etomoxir (CPT1 inhibitor) was injected at a final concentration of 400 mM before the injection of the above-described compounds. Basal respiration of cells treated with vehicle -Basal respiration of cells treated with etomoxir = basal respiration due to fatty acid oxidation; Basal respiration -Basal respiration due to fatty acid oxidation = basal respiration independent of fatty acid oxidation.
RNA sequencing and qPCR Total RNA was extracted with QIAzol Lysis Reagent (QIAGEN) and isolated using the RNeasy Plus Universal Mini Kit (QIAGEN). Library preparation and RNA sequencing were conducted by GATC Biotech AG on an Illumina platform with single-end 50 bp mRNA sequencing. Sequencing data were uploaded to the Galaxy platform  (Afgan et al. , 2016) , and usegalaxy. org server was.
ACKNOWLEDGMENTS We are grateful to  Tor Erik Rusten ,  Carl Thummel , and the  Developmental Studies Hybridoma Bank (DSHB)  for providing antibodies. We thank  Gabor Juhasz ,  Susan Abmayr ,  Zhe Han , the  Bloomington Drosophila Stock Center, FlyORF , and the  Vienna Drosophila RNAi Center (VDRC)  for providing flies. We thank  Christine Schmitt  and  Jacomina Krijnse Locker  from the  Ultrastructural BioImaging  facility of the  Pasteur Institute  for performing TEM; the  Cell Imaging platform, Structure Federative de Recherche de Necker, Inserm US 24  for assistance with microscopy; and  Ivan Nemazanyy  from the  Metabolism platform, Structure Federative de Recherche de Necker , for performing the Seahorse experiments. We are grateful to  Robin Klemm ,  Robert Ernst ,  Tobias Hermle , and  Philippe Chavrier  for critically reading the manuscript.
The work has been supported by the  ATIP-Avenir program  of the  Fondation Bettencourt-Schueller  ( Liliane Bettencourt Chair of Developmental Biology ) as well as state funding by the  Agence Nationale de la Recherche (ANR)  under the '' Investissements d'Avenir'' program  ( ANR-10-IAHU-01 ) and the  NEPHROFLY  ( ANR-14-ACHN-0013 ) grant to M. S. A. P. -M. is funded by a postdoctoral fellowship from the  Fondation pour la Recherche Medicale  ( FRM-SPF20170938629 ). This project has received funding from the  European Research Council (ERC)  under the European Union's  Horizon 2020 research and innovation program  grant agreement  804474  to  S. L. It  is also supported by the  Emmy Noether Programme  ( LI1817/2-1  to S. L. ) and Project  B07  of the collaborative research initiative ( SFB 1140  to S.
Here, we focus on these two themes-feeding biology diversity within the Hemiptera and Oncopeltus as a research model for macroevolutionary genetics. Key insights derive from a combination of global comparative genomics and detailed computational analyses that are supported by extensive manual curation, empirical data for gene expression, sequence validation, and new isoform-specific RNAi. We thereby identify genes with potentially restricted life history expression in Oncopeltus and that are unique to the Hemiptera, clarify evolutionary patterns of zinc finger protein family expansion, categorize predictors of insect gene structure, and identify lateral gene transfer and amino acid metabolism features that correlate with feeding biology.
Results and discussion The genome and its assembly Oncopeltus fasciatus has a diploid chromosome number (2n) of 16, comprised of seven autosomal pairs and two sex chromosomes with the XX/XY sex determination system  [28, 29]. To analyze this genetic resource, we sequenced and assembled the genome using next-generation sequencing approaches (Table  1 , see also the "Methods" section and Additional file 1: Supplemental Notes Sections 1-4). We measure the genome size to be 923 Mb in females and 928 Mb in males based on flow cytometry data (Additional file 1: Supplemental Note 2. 1. a). The assembly thus contains 84% of the expected sequence, which is comparable to other recent, medium-sized insect genomes  [12, 30]. However, our analyses of the k-mer frequency distribution in raw sequencing reads yielded ambiguous estimates of genome size and heterozygosity rate, which is suggestive of high heterozygosity and repetitive content (  [31] , Additional file 1: Supplemental Note 2.
Gene expression profiles across the milkweed bug life cycle To augment published transcriptomic resources  [35, 36] , we sequenced three different post-embryonic samples ("i5K" dataset, see the "Methods" section). We then compared the OGS to the resulting de novo transcriptome and to a previously published embryonic and maternal (ovary) transcriptome ("454" pyrosequencing dataset,  [35] ). Our OGS is quite comprehensive, containing 90% of transcripts from each transcriptomic dataset (Fig. 2a ). The OGS also contains an additional 3146 models (16% of OGS) not represented in either transcriptome, including 163 de novo models encoding chemoreceptors. Such genes are known for lineage-specific expansions and highly tissue-and stage-specific expression (  [37, 38] , and see below), and our OGS captures these genes with rare transcripts.
2b ). The inclusion of a fifth dataset from a published adult library  [36]  provided only a 1% gain in expression support, indicating that with the current study the expression data volume for Oncopeltus is quite complete. RNA-seq studies were further conducted to establish male-, female-, and nymph-specific gene sets (Fig. 2b, c , Additional file 1: Supplemental Note 2. 4), from which we also infer that the published adult dataset of unspecified sex is probably male. Moreover, most genes with stage-restricted or stage-enriched expression are in our male sample (Fig. 2b, c ). For example, gustatory receptor (GR) genes show noticeable restriction to the adult male and published adult (probable male) samples (n = 169 GRs: 40% no expression, 27% only expressed in these two samples), with half of these expressed in both biological replicates (52%).
Interestingly, the nymphal sample is enriched for genes encoding structural cuticular proteins (94%, which is > 56% more than any other sample). This likely reflects the ongoing molting cycles, with their cyclical upregulation of chitin metabolism and cuticular gene synthesis  [40] , that are experienced by the different instars and molt cycle stages of individuals pooled in this sample. Lastly, gene sets with sex-specific Fig. 2  Comparisons of the official gene set and transcriptomic resources for Oncopeltus fasciatus. a Area-proportional Venn diagram comparing the OGS v1. 1 (OGS), a Trinity de novo transcriptome from the three post-embryonic RNA-seq samples (i5K) and the maternal and embryonic transcriptome from 454 data ("454",  [35] ). Sample sizes and the fraction of each transcriptome represented in the OGS are indicated (for the 454 dataset, only transcripts with homology identification were considered).
The unique fraction of each set is also specified (%). Dataset overlaps were determined by blastn (best hit only, e value < 10 -9 ). b Venn diagram of gene model expression support across four life history samples. Values are numbers of gene models, with percentages also given for the largest subsets. Note that the "Embryo/Maternal" sample derives from 454 pyrosequencing data and therefore has a smaller data volume than the other, Illumina-based samples. c Summary of sex-and developmental stage-specific RNA-seq comparisons across hemipteroid species: Apis, Acyrthosiphon pisum; Clec, Cimex lectularius; Focc, Frankliniella occidentalis (thysanopteran outgroup); Ofas, Oncopeltus fasciatus; Pven, Pachypsylla venusta; n. d. , not determined. For complete numerical details, see Additional file 1: Supplemental Note 2.
Protein orthology and hemipteran copy number comparisons To further assay protein-coding gene content, we compared Oncopeltus with other arthropods. A phylogeny based on strictly conserved single-copy orthologs correctly reconstructs the hemipteran and holometabolan clades' topologies (Fig. 3a , compare with Fig. 1a ), although larger-scale insect relationships remain challenging  [3]. We then expanded our appraisal to the Benchmarking Universal Single-Copy Orthologs dataset of 1658 Insecta genes (BUSCO v3,  [42] ). Virtually all BUSCO genes are present in the Oncopeltus OGS (98. 9%, Fig. 3b , Additional file 1: Supplemental Note 6. 1). Although some genes are fragmented, the assembly has a high level of BUSCO completeness (94. 6%), independent of the annotation prediction limitations that missed some exons from current gene models.
We identified 762 putative TFs in Oncopeltus, which is similar to other insects for total TF count and for the size of each TF family (Fig. 4a : note that the heatmap also reflects the large, duplicated repertoire in the pea aphid, see also Additional file 2: Tables S6. 3-S6. 5). We were able to infer DNA binding motifs for 25% (n = 189) of Oncopeltus TFs, mostly based on data from Drosophila melanogaster (121 TFs) but also from distantly related taxa such as mammals (56 TFs). Such high conservation is further reflected in explicit orthology assignments for most proteins within several large TF families, including the homeodomain (53 of 85, 62%), basic helix-loop-helix (bHLH, 35 of 45, 78%), and forkhead box (16 of 17, 94%) families. In contrast, most C2H2 zinc finger proteins lack orthology assignment (only 22 of 360, 6%).
Overall, we find the TF pipeline results to be a robust and reasonably comprehensive representation of these gene classes in Oncopeltus. These analyses also uncovered a correction to the published Oncopeltus literature for the developmental patterning proteins encoded by the paralogs engrailed and invected. These genes arose from an ancient tandem duplication prior to the hexapod radiation. Their tail-to-tail orientation enables ongoing gene conversion  [58] , making orthology discrimination particularly challenging. For Oncopeltus, we find that the genes also occur in a tail-to-tail orientation and that invected retains a diagnostic alternative exon  [58]. These new data reveal that the purported Oncopeltus engrailed ortholog in previous developmental studies (e. g. ,  [54, [59] [60] [61] [62] ) is in fact invected (Additional file 1: Supplemental Note 5.
), and Diptera (Dipt. ). The dashed line demarcates the dipteran family Culicidae (mosquitoes). c Proportions of Oncopeltus homeodomain (HD) and C2H2 zinc finger proteins with orthology assignment (predicted DNA binding specificity) and/or manual curation. "Classified" refers to the automated classification of a protein to a TF family, but without a specific orthology assignment. d Maximum likelihood phylogeny of representative subsets of the zinc finger 271-like family in Oncopeltus (49 proteins, blue text) and the pea aphid (55 proteins, black text), with chelicerate (red text) and holometabolan (yellow text) outgroups (16 proteins, 7 species), based on the Oncopeltus OGS and GenBank protein accessions. Gaps were removed during sequence alignment curation; all nodes have ≥ 50% support; branch length unit is substitutions per site  [157].
5a , with the sole exception of the honey bee), and we therefore used our gap-filled dataset as a more accurate basis for further comparisons. To support direct comparisons among hemipterans, we also performed our RepeatModeler analysis on the bed bug and pea aphid assemblies. Repeats comprised 36% and 31% of the respective assemblies, similar to the gap-filled value of 32% in Oncopeltus. Nevertheless, given the smaller sizes of these species' assemblies-651 Mb in the bed bug and 542 Mb in the pea aphid-the absolute repeat content is much higher in Oncopeltus (Fig. 5b ). Excluding unknown repeats, the most abundant transposable elements in Oncopeltus are LINE retrotransposons, covering 10% of the assembly (Additional file 2: Table  S2. 5). This is also the case in the bed bug (12%), while in the pea aphid DNA transposons with terminal inverted repeats (TIRs) are the most abundant (2% of the assembly identified here and 4% reported from manual curation in the pea aphid genome paper,  [6] ).
Lineage-and genome size-related trends in insect gene structure Both our manual curation work and BUSCO analyses highlighted the fact that Oncopeltus genes are often comprised of many, small exons. We thus undertook a comparative analysis to determine whether this is a general feature to be considered for structural annotation of hemipteran genomes. We find that both lineage and genome size can serve as predictors of gene structure. Firstly, we created a high-quality dataset of 30 functionally diverse, large genes whose manual curation could reasonably ensure complete gene models across 7 species from 4 insect orders (Fig. 6a , Additional file 1: Supplemental Note 6. 3). Most species encode the same total number of amino acids for these conserved proteins, with the thrips Frankliniella occidentalis and the fruit fly being notable exceptions with larger proteins (Fig.
This large dataset corroborates our original findings, with bugs having small exons while both the median and Q3 quartile reflect larger exon sizes in beetles and flies (Fig. 6b ). Notably, the median and median absolute deviation are highly similar between species pairs within the Hemiptera and Coleoptera. Meanwhile, the exon metrics within the Diptera support large protein sizes as a drosophilid-specific, rather than dipteran-wide, feature. Where it was not possible to analyze all 30 genes for a given species, equal sampling was done across the range of protein sizes of the complete dataset, based on the Cimex ortholog sizes (1:1:1 sampling from big-to-medium-to-small subcategories of 10 genes each). b Box plot representations of coding sequence exon size (aa) for 2 species from each of 3 insect orders, based on datasets of unique coding sequence exons (1 isoform per gene) and excluding terminal exons < 10 aa (as most of those exons may rather be UTRs or a small placeholder N-terminal exon based on automated Maker model predictions).
It will be interesting to see if the correlation with genome size is borne out in other invertebrate taxa. Species are represented by their four-letter abbreviations (as in Fig. 6 ), and estimated genome sizes are indicated parenthetically (measured size  [12, 30, 162, 165, 166] ; draft assembly size: GenBank Genome IDs 14741 and 17730). Divergence times are shown in gray and given in millions of years  [3]. Abbreviations as in Figs. 4  and 6 , and also: Hemipt. , hemipteroid assemblage (including F. occidentalis); n. d. , no data The selective pressures and mechanisms of intron gain in the Hemiptera will be a challenge to uncover. While median exon size (Fig. 6b ) could reflect species-specific nucleosome sizes  [75, 76] , this does not explain why only the Hemiptera seldom exceed this (Fig.
We therefore used the CycADS annotation pipeline  [113]  to reconstruct the Oncopeltus metabolic network. The resulting BioCyc metabolism database for Oncopeltus (OncfaCyc) was then compared with those for 26 other insect species (  [114] , http://arthropodacyc. cycadsys. org/), including 3 other hemipterans: the pea aphid, the green peach aphid, and the kissing bug (Tables  3  and 4 ). For a global metabolism analysis, we detected the presence of 1085 Enzyme Commission (EC) annotated reactions with at least 1 protein in the Oncopeltus genome (Additional file 1: Supplemental Note 6. 4, Additional file 2: Table  S6. 10). Among these, 10 enzyme classes (represented by 17 genes) are unique and 17 are missing when compared to the other insects (Table  4 , Additional file 2: Table  S6. 11). We then specifically compared amino acid metabolism in the four hemipterans representing the three different diets.
Functional testing will clarify the roles of genes we have identified as unique to the Hemiptera, including those implicated in chemical protection, bacterial and plant cell wall metabolism, or encoding wholly novel proteins (Figs. 3  and 8 , see also Additional file 1: Supplemental Note 2. 2). Meanwhile, the prominent and species-specific expansions specifically of ZF271-like zinc fingers (Fig. 4 ), combined with the absence of the co-repressor KAP-1 in insects, argues for investigation into alternative interaction partners, which could clarify the nature of these zinc fingers' regulatory roles and their binding targets. One key output of this study is the generation of a metabolism database for Oncopeltus, contributing to the ArthropodaCyc collection (Table  3 ). In addition to comparisons with other species (Fig.
10 ), this database can also serve as a future reference for studies that use Oncopeltus as an ecotoxicology model (e. g. ,  [121] ). While we have primarily focused on feeding ecology in terms of broad comparisons between phytophagy and fluid feeding, Oncopeltus is also poised to support future work on nuances among phytophagous species. Despite its milkweed diet in the wild, the lab strain of Oncopeltus has long been adapted to feed on sunflower seeds, demonstrating a latent capacity for more generalist phytophagy  [112]. This potential may also be reflected in a larger gustatory receptor repertoire than would be expected for  an obligate specialist feeder (Table  2 ). Thus, Oncopeltus can serve as a reference species for promiscuously phytophagous pests such as the stink bug. Finally, we have identified a number of key genes implicated in life history trade-offs in Oncopeltus, for traits such as cardenolide tolerance, pigmentation, and plasticity in reproduction under environmental variation.
Transcriptome resources Total RNA from three distinct life history samples (pooled, mixed-instar nymphs; an adult male; an adult female) was also sequenced on an Illumina HiSeq2000s machine, producing a total of 72 million 100-bp paired-end reads (Additional file 1: Supplemental Note 1. 3, Additional file 2: Table  S1. 1; GenBank Bioproject: PRJNA275739). These expression data were used to support the generation of the OGS at different stages of the project: as input for the evidence-guided automated annotation with Maker 2. 0 (Additional file 1: Supplemental Note 3), as expression evidence tracks in the Apollo browser to support the community curation of the OGS, and, once assembled into a de novo transcriptome, as a point of comparison for quality control of the OGS. The raw RNA-seq reads were pre-processed by filtering out low-quality bases (phred score < 30) and Truseq adapters with Trimmomatic-0.
30. Further filtering removed ribosomal and mitochondrial RNA sequences with Bowtie 2  [138] , based on a custom library built with all hemipteran ribosomal and mitochondrial RNA accessions from NCBI as of 7th February 2014 (6069 accessions). The pooled, filtered reads were mapped to the genome assembly with Tophat2-PE on CyVerse  [139]. A second set of RNA-seq reads from an earlier study ("published adult" dataset,  [36] ) was also filtered and mapped in the same fashion, and both datasets were loaded into the Oncopeltus Apollo instance as evidence tracks (under the track names "pooled RNA-seq -cleaned reads" and "RNA-seq raw PE reads Andolfatto et al", respectively). Additionally, a de novo transcriptome was generated from our filtered RNA-seq reads (pooled from all three samples prepared in this study) using Trinity  [140]  and TransDecoder  [141]  with default parameters.
This transcriptome is referred to as "i5K," to distinguish it from a previously published maternal and early embryonic transcriptome for Oncopeltus (referred to as "454",  [35] ). Both the i5K and 454 transcriptomes were mapped to the genome assembly with GMAP v. 2014-05-15 on CyVerse. These datasets were also loaded into the Apollo browser as evidence tracks to assist in manual curation.
Life history stage-specific and sex-specific expression analyses in hemipteroids Transcript expression of the OGS v1. 1 genes was estimated by running RSEM2  [142]  on the filtered RNA-seq datasets for the three i5K postembryonic stages against the OGS v1. 1 cDNA dataset. Transcript expression was then based on the transcripts per million (TPM) value. The TPM values were processed by adding a value of 1 (to avoid zeros) and then performing a log2 transformation. The number of expressed genes per RNA-seq library was compared for TPM cutoffs of > 1, > 0. 5, and > 0. 25. A > 0. 25 cutoff was chosen, which reduced the number of expressed genes by 6. 6% compared to a preliminary analysis based on a simple cutoff of ≥ 10 mapped reads per transcript, while the other TPM cutoffs were deemed too restrictive (reducing the expressed gene set by > 10%).
This analysis was also applied to the "published adult" dataset  [36]. To include the embryonic stages in the comparison, transcripts from the 454 transcriptome were used as blastn queries against the OGS v1. 1 cDNA dataset (cutoff e value < 10 -5 ). The results from all datasets were converted to a binary format to generate Venn diagrams (Fig. 2b ). Statistically significant sex-specific and developmental stage-specific gene enrichment was determined from RNA-seq datasets according to published methods  [143, 144] , with modifications. Data from Oncopeltus (see the previous methods section, Bioproject: PRJNA275739) were compared between stages and pairwise with the hemipterans Cimex lectularius, PRJNA275741; Acyrthosiphon pisum, PRJNA209321; and Pachypsylla venusta, PRJNA275248; as well as with the hemipteroid Frankliniella occidentalis (Thysanoptera), PRJNA203209 (see also Fig.