Aspergillus and Mucorales is very rare and is usually associated in immunocompromised patients with a high mortality rate. This rare co-infection leads to difficulties in diagnosis, and therapeutic delays can result in a poor prognosis. Overall, the treatment of choice is surgical debridement and liposomal amphotericin B. This paper describes a combined aspergillosis and mucormycosis case in a diabetes mellitus type 2 patient with chronic ulcerations of the palatal and cheek. To our knowledge, this is the first report of an uncommon co-infection of Aspergillus fumigatus and Rhizopus arrhizus in a rhino-orbital presentation.Aspergillosis and mucormycosis are filamentous fungal infections occurring predominantly in immunocompromised patients. Fulminant process with rapid infiltration of the contiguous tissue is distinctive for both type of fungi. The rhinocerebral co-infection by  Aspergillus genus; A. fumigatus is the most important pathogenic fungus in humans; however, other species had been associated in different pathologies. Some clinical presentations are often similar among A. fumigatus related species, and are frequently misidentified [Mucormycosis and invasive aspergillosis infections are aggressive fungal infections with a high mortality rate. These infections have generally predilection among patients with risk factors as immunosuppression, untreated diabetes mellitus, kidney diseases, hematological malignancies or mayor trauma ,2,3. Aspentified ,5.Rhizopus, Mucor and Lichtheimia are the main genera implicated as cause of mucormycosis [Rhizopus is the etiological agent of 70% of the reported cases [Following aspergillosis, mucormycosis is the second most frequently mycosis caused by filamentous fungi. Agents of mucormycosis belong to the order Mucorales. rmycosis . The mosed cases ,7. Reports of combined mucormycosis and aspergillosis infections limited to the oral-rhinocerebral region are exceptional. Diagnosis is often based on histopatological examination and supported by fungal isolation. The peculiarity of this infection leads to difficulties in diagnosis and delays can result in a poor prognosis ,9. This A 52-year-old male patient with a history of diabetes mellitus type 2 with apparently regular control was presented to the emergency room in January of 2016 with a 2-month history of swelling of the left side of the face, left facial palsy, ptosis, and an impaired vision. At clinical examination, there was prominent tumefaction of the left hemi-face, involving the peri-orbital, zygomatic and maxillary areas, naso-sinusal congestion and diffuse ulcerative and necrotic lesion of the palatal and cheek mucosa A,B. The This study was approved by the Institutional Review Board of Hospital General Dr. Manuel Gea Gonzalez, Mexico City, Mexico .Since the mycological culture was negative, it was decided to carry out the molecular identification in order to identify the causal agents associated with the patient. In this way, two paraffin-embedded tissue samples were analyzed, one sample from naso-sinus area and the second sample from maxillar sinus. The genomic DNA was isolated using a DNeasy blood and tissue kit  according to the manufacturer’s instructions after preliminary removal of paraffin by extraction with xylene protocol. Molecular identification was performed by ITS rDNA region and beta-tubulin gene amplification and sequencing ,11,12. FFor beta-tubulin gene amplification primers (5′-AATTGGTGCCGCTTTCTGG-3′) and (5′-AGTTGTCGGGACGGAATAG-3′) were used . PCR reahttps://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 9 December 2021). using BLASTn analysis and displayed 98.65% of homology with A. fumigatus ATCC 1022, A. oerlinghausenensis CBS139183 (98.85%), A. fumisynnematus IFM42277 (99.80%), A. lentulus NRRL 35552 (99.81%), A. novofumigatus CBS 117250 (99.61%), A. fumigatiaffinis CBS 117186 (99.42%), A. fischeri NRLL 181 (99.23%), A. laciniosus KACC 41657 (99.23%), A. spinosus NRRL 5034 (99.23%), A. takakii IFM 53599 (99.23%), Aspergillus clavatus ATCC 1007 95.42% and, A. cervinus NRRL 5025 (93.15%), among others species from naso-sinusal sample and 100% of homology with Rhizopus arrhizus (syn. R. oryzae) HP25 and 8–3 M strains, among others from maxillar samples [For ITS rDNA region amplification, a 517 bp product was amplified from naso-sinusal sample, and 620 bp product from maxillar sample. The PCR products were purified and the nucleotide sequence was determined in both directions using the same primers with Taq FS dye terminator cycle sequencing fluorescence-based sequencing and analyzed on an applied biosystems 3730 xl DNA sequencing system. The consensus sequences homology search was performed with ex-type ITS rRNA fungi databases . A 485 bp fragment from naso-sinusal and maxillar sample was amplified and the nucleotide sequence was determined in both directions using the same primers. The sequences homology search was performed with ex-type strains sequences under section Aspergillus subgen. Fumigati using BLASTn analysis The sequence showed a 98.74% of homology with A. fumigatus ATCC 1022, A. oerlinghausenensis CBS139183 (94.20%), A. fischeri NRLL 181 (93.08%), A. spinosus NRRL 5034 (91.84%), A. laciniosus KACC 41657 (91.18%), A. takakii IFM 53599 (91.08%), A. lentulus NRRL 35552 (90.59%), A. fumigatiaffinis CBS 117186 (90.38%), A. novofumigatus CBS 117250 (89.98%), A. fumisynnematus IFM42277 (89.25%), Aspergillus clavatus ATCC 1007 (78.10%) and, A. cervinus NRRL 5025 (77.57%) [Rhizopus arrhizus (syn. R. oryzae) GEA-44, Aspergillus fumigatus GEA-44, and Aspergillus fumigatus GEA-1 for benA gene, sequences were deposited in the GenBank under accession number MG029627, MT340910 and MT370439, respectively.The ITS rDNA region does not allow species-level discrimination for the genus A. fumigatus, followed by A. terreus, A. niger, A. flavus, and other species belonging to Fumigati section [A. lentulus has been described recently as a new species of Aspergillus genus; genetic analysis indicated its close relation with A. fumigatus, and it has also been reported as a cause of invasive aspergillosis [Aspergillus is a challenge for both clinical microbiology and mycology laboratories, which complicates epidemiological investigations leading often to its misidentification as an atypical A. fumigatus. Aspergillosis and mucormycosis are filamentous fungal infections occurring predominantly in immunocompromised patients, and both types of infection are characterized for being fulminant processes with rapid infiltration of contiguous tissues. The most common cause of invasive aspergillosis is  section . A. lentgillosis ,12. The The classical fungi diagnosis involves a rapid microscopic identification using a KOH/chlorazol black preparation. The mycological culture is frequently reported as negative in half of the cases of mucormycosis. One of the main causes is the homogenization of the tissue since it can generate the loss of viability of the non-separated fragile hyphae of these fungi. In our clinical case, we failed at least three times in the mycological culture and one of the reasons was that we homogenized the tissue prior to culture. Therefore, it is recommended to slice and cut the tissue into very small and thin pieces before inoculation in the culture medium. Another problem associated with negative results is the use of a single culture medium (Sabouraud agar), so it is recommended to use other culture methods such as potato dextrose agar, Malt extract agar and Brain–heart–infusion (BHI) broth. The use of BHI broth provides optimal contact between the medium and the sample. In addition, growing at two temperatures (30 °C and 37 °C) can also increase yield of culture. Culture requires at least three days, and it needs mycology expertise for fungal description ,15.Aspergillus; however, accurate and definitive identification is achieved by molecular methods [Aspergillus. For the molecular identification of Aspergillus fumigatus, experts recommend comparative sequence analysis of the ribosomal ITS region, specifically the ITS1 and ITS2 flanking regions of the 5.8S rDNA for the identification of the Aspergillus Fumigati section (intersection) and of the beta-tubulin or calmodulin genes for the identification of species level (intrasection). In this study, analysis of ITS rRNA region combined with amplification of benA gene, allowed the identification of the two etiologic fungal agents [Histopathological examination displays characteristic fungal structures, such as large non-septate hyphae for Mucorales and septated branching hyphaes for  methods ,11,16. Cl agents . The corBoth mucormycosis and aspergillosis are usually severe infections, with mortality rates ranging from 20% to 40% in immune-compromised patients and approaching to 80–90% in those cases of disseminated infections or involvement of the brain structures ,8. ApproA. fumigatus and R.arrhizus infection. Herein, to our knowledge, this is the first case of rhino-obital infection due to this rare fungal combination.Our case presented a rhino-orbital compromise, associated with diabetes mellitus, the most prevalent underlying disease, and was treated with amphotericin B, which is considered as a fundamental backbone of treatment for both agents; we also aimed to identify a mixed


Objective: Pancreatic cancer is a highly lethal malignancy globally. This study aimed to probe and validate immune-related prognostic mRNAs as therapeutic targets for pancreatic cancer.Methods: Gene transcriptome data of pancreatic cancer and normal pancreas were retrieved from TCGA-GTEx projects. Two thousand four hundred and ninety-eight immune-related genes were obtained from the IMMUPORT database. Abnormally expressed immune-related genes were then identified. Under univariate and multivariate cox models, a gene signature was constructed. Its predictive efficacy was assessed via ROCs. The interactions between the 21 genes were analyzed by Spearson analysis and PPI network. Using the GEPIA and The Human Protein Atlas databases, their expression and prognostic value were evaluated. The TIMER database was utilized to determine the relationships between MET, OAS1, and OASL mRNAs and immune infiltrates. Finally, their mRNA expression was externally verified in the GSE15471 and GSE62452 datasets.Results: An immune-related 21-gene signature was developed for predicting patients' prognosis. Following verification, this signature exhibited the well predictive performance. There were physical and functional interactions between them. MET, OAS1, and OASL mRNAs were all up-regulated in pancreatic cancer and associated with unfavorable prognosis. They showed strong correlations with tumor progression. Furthermore, the three mRNAs were distinctly associated with immune infiltrates. Their up-regulation was confirmed in the two external datasets.Conclusion: These findings identified three immune-related prognostic mRNAs MET, OAS1, and OASL, which may assist clinicians to choose targets for immunotherapy and make personalized treatment strategy for pancreatic cancer patients. Pancreatic cancer is a highly lethal disease with fairly high mortality globally . Annual High-throughput sequencing may assist us to probe therapeutic targets for cancer therapies and understand the underlying mechanisms of the anti-cancer efficacy in depth . Pancreahttps://xenabrowser.net/datapages/). Clinical information of these pancreatic cancer patients including gender, pathologic T, N and histologic grade was retrieved from Genomic Data Commons (GDC). Those without available follow-up data were removed. Gene transcriptome data of 165 normal pancreas samples from the Genotype-Tissue Expression (GTEx) project and 178 pancreatic cancer samples from The Cancer Genome Atlas (TCGA) database were downloaded based on the UCSC Xena browser (https://www.ncbi.nlm.nih.gov/gds/). Of which, the GSE15471 dataset included 39 pairs of pancreatic cancer and normal tissues. Meanwhile, the GSE62452 dataset contained 69 pancreatic cancer and adjacent normal tissue specimens (Two microarray datasets GSE15471 and GSE62452 were downloaded from the Gene Expression Omnibus (GEO) database (pecimens .via the Linear Models for Microarray Data (limma) package in R (https://www.immport.org/home). Following integration of DEGs and immune-related genes, abnormally expressed immune-related genes were identified for pancreatic cancer.After merging gene matrix of GTEx and TCGA datasets based on the Ensembl IDs, differentially expressed genes (DEGs) between pancreatic cancer and normal pancreas specimens were screened age in R . The scrp < 0.001 were considered related to pancreatic cancer prognosis. Then, candidate genes were screened via multivariate Cox regression analysis. Based on them, a risk score was established according to the following formula: risk score = β1x1 + β2x2 + … + βixi . The risk score of each patient was calculated and all patients were separated into high- and low-risk groups in accordance with the median value. Kaplan–Meier survival analysis was conducted between high- and low-risk groups through the survival package in R. The Receiver Operating Characteristic curves (ROCs) for overall survival were drawn utilizing the survivalROC package in R  including biological processes (BP), molecular functions (MF) and cellular components (CC) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analyses of the survival-related genes were presented for exploring underlying biological functions age in R . Terms wvia the Search Tool for the Retrieval of Interacting Genes Database (STRING) database (http://string-db.org/)  . The degvia the online GEPIA database (http://gepia.cancer-pku.cn/index.html)  . Furtherhttps://www.proteinatlas.org/). Furthermore, their immunofluorescence images were also downloaded.Immunohistochemistry of MET, OAS1, and OASL proteins in pancreatic cancer and normal pancreas specimens was obtained from The Human Protein Atlas  of MET, OAS1, and OASL and the abundance of immune infiltrates composed of B cell, CD4o/timer/ ).P < 0.05 indicated statistical significance.All analyses were presented utilizing R version 3.5.2. Spearman correlation analysis was used to assess the associations between genes from the gene signature. The strengths of correlations were determined as follows: 0–0.39: weak; 0.40–0.59: moderate; 0.60–0.79: strong; 0.80–1.0: very strong. The differences in gene expression between two subgroups were calculated using the Wilcox test. n = 178) than normal pancreas specimens (n = 165), which were composed of 962 down- and 775 up-regulated genes listed in p = 1.499e-14; From TCGA-GTEx datasets, 1,737 DEGs with |log FC| > 2 and FDR <0.05 were identified for pancreatic cancer : 2.180 (1.283–3.706), p = 0.004] and risk score  were risk factors for pancreatic cancer . Except for IL22RA1 and PAK3, most of them were up-regulated in pancreatic cancer. We further calculated their correlations at the expression levels using Spearson analysis, as shown in In p = 0.00044; p = 0.00023; p = 0.047; p = 0.0072; p = 0.005; p = 0.012; p = 0.019; p = 0.006; p = 0.048; p = 0.009; Among 21 genes from the immune-related signature, MET, OAS1, and OASL were significantly associated with prognosis of pancreatic cancer patients. Patients with high MET expression were indicative of shorter DFS (p = 2.08e-02), CD8 + T cell , CD4 + T cell , neutrophil , and dendritic cell  in p = 5.89e-05) and dendritic cell  that were all up-regulated in pancreatic cancer and indicated an unfavorable prognosis. Following multiple dataset verification, the three genes might be promising therapeutic targets, which were worthy of further research.Despite the TNM stage system as an efficient tool to detect tumor stage, there is discrepancy in prognosis based on TNM stage . Thus, tThe molecular mechanisms of highly aggressive behaviors remain unknown. We analyzed biological functions of survival-related genes. These genes could regulate migration of multiple cells such as epithelial and endothelial cells and were involved in key cellular components like endoplasmic reticulum lumen and specific granule and possess different molecular functions such as receptor activity, cytokine binding, and growth factor activity. These data were indicative that these genes were involved in tumor progression. For example, CCN1/Cyr61 secreted by pancreatic cancer cells may promote migration of endothelial cells . Nerve gOur study revealed that there were physical and functional interactions between 21 genes from the immune-related gene signature. Among them, MET, OAS1, and OASL were verified to be markedly up-regulated in pancreatic cancer and associated with poor clinical outcomes of patients. Furthermore, MET expression was significantly correlated to infiltration of B cell, CD8 + T cell, CD4 + T cell, neutrophil, and dendritic cell. MET gene is located on chromosome 7q21-31. Changes in MET functions have been a hallmark of multiple cancers including pancreatic cancer . MET ovein vitro experiments. Second, their prognostic values should be verified in prospective research.Collectively, we identified three immune-related prognostic genes MET, OAS1, and OASL, which could be promising therapeutic targets as well as prognostic markers for pancreatic cancer. However, several limitations of this study should be pointed out. First, the biological functions of MET, OAS1, and OASL such as their interactions with immune cells in tumor microenvironment need to be explored In this study, we established an immune-related 21-gene signature for prediction of pancreatic cancer prognosis. This signature could be accurately and independently predictive of patients' survival. Among these genes, MET, OAS1, and OASL were validated to be up-regulated in pancreatic cancer and associated with unfavorable prognosis of patients. Also, there were closely interactions between them and immune infiltrates. Thus, MET, OAS1, and OASL could be potential therapeutic markers in pancreatic cancer.The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/HL conceived and designed the study. CZ, FN, and PH conducted most of the experiments, data analysis, and wrote the manuscript. YZo, YZh, YL, and HF participated in collecting data and helped to draft the manuscript. All authors reviewed and approved the manuscript.YZh was employed by the company Jiangsu ALF Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Handling Editor declared a shared affiliation, though no other collaboration, with one of the authors CZ.


Caenorhabditis elegans. Transthyretin-like proteins were the only component common to all adult secretomes; many other protein classes overlapped across multiple datasets. The glycolytic enzymes aldolase and enolase were present in all parasitic species, but missing from C. elegans. Secretomes from larval stages showed less overlap between species. Although comparison of secretome composition across species and life-cycle stages is challenged by the use of different methods and depths of sequencing among studies, our workflow enabled the identification of conserved protein families and pinpointed elements that may have evolved as to enable parasitism. This strategy, extended to more secretomes, may be exploited to prioritize therapeutic targets in the future.Parasitic nematodes are highly successful pathogens, inflicting disease on humans, animals and plants. Despite great differences in their life cycles, host preference and transmission modes, these parasites share a common capacity to manipulate their host’s immune system. This is at least partly achieved through the release of excretory/secretory proteins, the most well-characterized component of nematode secretomes, that are comprised of functionally diverse molecules. In this work, we analyzed published protein secretomes of parasitic nematodes to identify common patterns as well as species-specific traits. The 20 selected organisms span 4 nematode clades, including plant pathogens, animal parasites, and the free-living species  Caenorhabditis elegans. This was the case of two glycolytic enzymes, aldolase and enolase, for which alternative functions have been proposed by others. A role in parasitic processes is intriguing. Our workflow enabled the identification of conserved protein families and pinpointed elements that may have evolved as to enable parasitism. This may prove useful in the future to identify and prioritize potential targets for therapeutic interventions.Parasitic helminths (worms) cause long-lasting infections. In order to survive in their hosts, this class of pathogens has developed various strategies; one of them consists of releasing soluble mediators  proteins), which dampen the host immune response. Here, we analyzed and compared published ES protein catalogs of parasitic nematodes to identify common patterns as well as species-specific traits. Many proteins were common to multiple species, and a few were absent in secretions from the non-parasitic species  Nematodes are a ubiquitous and highly diverse group of organisms. A parasitic lifestyle has evolved at least 15 times in this phylum, with multiple species becoming reliant on plant or animal hosts for survival and reproduction [Ascaris lumbricoides, Trichuris trichiura, and the hookworms), mostly in tropical and subtropical areas of the world [Parasitic species cause debilitating pathologies and immense economic losses. In 2013, 1.75 billion humans were estimated to be infected with the major parasitic nematode species of the gastrointestinal (GI) tract (he world .Wuchereria bancrofti and Brugia spp.) can persist for at least 6–8 years [Onchocerca volvulus, this can be 10–15 years (retrieved from https://www.cdc.gov/dpdx/onchocerciasis/index.html). Despite great differences in life cycles, preferential hosts and transmission modes, parasitic nematodes share a common capacity to manipulate their host’s immune system. Their immunomodulatory properties explain at least in part their longevity in mammalian hosts [One peculiarity of nematode and other helminth infections is their typically long-lasting, chronic nature. In mammalian hosts, adult worms can live for several months to many years. For instance, filarial nematodes causing lymphatic filariasis  products from a number of nematode species have been at least partially characterized biochemically. ES products are described as both substances that are actively secreted by helminths and products that are released during physiological processes such as digestion or egg-laying [Nematode infections typically induce a regulatory, “Th2-type” immune response in mammals, involving CD4+ regulatory T cells, regulatory B cells, alternatively activated macrophages, and type 2 cytokines, among other effectors of the innate and adaptive arms of type 2 immunity ,8. A halg-laying ,10. Hencg-laying ,11. Excrg-laying ,13, (ii)g-laying , (iii) dg-laying ,15, or ig-laying .Necator americanus ESP , which have shown different degrees of success [Acanthocheilonema viteae, which are mediated by post-translational modification of this protein with phosphorylcholine, may lead to treatments for various inflammatory conditions [The attractiveness of the immunomodulatory and anti-inflammatory properties of nematode ESP for drug or vaccine design has been acknowledged for many years. Promising vaccine candidates destined for use in humans or animals have been taken to early phases of clinical development . For exa success . Similarnditions –22.Caenorhabditis elegans to highlight ESP components that may be associated with parasitism.In this study, we gathered published protein secretomes of multiple species of parasitic nematodes to identify common patterns as well as species-specific protein secretion traits. These organisms span 4 nematode clades, including 3 plant and 16 animal parasites. Among parasites of mammals, 12 occupy the GI tract of their hosts, one is located in blood vessels, one resides in lymph nodes, and 2 inhabit other tissues. They display a wide variety of preferential hosts and life histories. Finally, we included previously unpublished secretome data from the non-parasitic free-living nematode Caenorhabditis elegans N2 strain was maintained under standard conditions on agar plates with Escherichia coli OP50 as food source [g for 2 min. A cocktail of protease inhibitors  was added to the recovered media, which was then passed through a 0.22 μm filter. Sample concentration and further processing was performed as previously described [m/z. MS/MS scans were acquired from 50 to 1,990 m/z, scan time was 1.35 s, and the interscan interval was 0.15 s. Doubly and triply charged ions were selected for fragmentation with collision energies calculated using a linear curve from reference collision energies.d source . L4 –youescribed . ESP mixescribed . The enthttp://www.matrixscience.com/distiller.html) with peak picking parameters set at 5 for Signal Noise Ratio (SNR) and at 0.4 for Correlation Threshold (CT). Briefly, MS/MS peak lists (MGF files) generated were analyzed using Mascot  and X! Tandem (Version: 2007.01.01.1) [C. elegans and Escherichia coli proteins in the Uniprot database . To rank the proteins based on absolute abundance, the emPAI spectrum counting method was employed in Scaffold [10.17632/727hjmhwn5.1; https://data.mendeley.com/datasets/727hjmhwn5/1).MS raw data from a single run were acquired on the Data Directed Analysis feature in the MassLynx (Micromass) software with a 1, 2, 4 duty cycle . MS/MS raw data were transferred from the Q-TOF Microcomputer to a server and automatically manipulated for generation of peaklists by employing Distiller version 2.3.2.0 (01.01.1) ,26. Scaf01.01.1) . PeptideScaffold . Data frScaffold  to generScaffold ,31 and SScaffold . The datHaemonchus contortus [Ancylostoma caninum [Heligmosomoides polygyrus bakeri [Nippostrongylus brasiliensis [Necator americanus [Strongyloides ratti [Strongyloides venezuelensis [Ascaris suum [Dirofilaria immitis [Brugia malayi [Onchocerca ochengi [Litomosoides sigmodontis [Spirocerca lupi [Gnathostoma spinigerum [Trichuris muris [Trichuris suis [Meloidogyne incognita [Bursaphelenchus xylophilus [Bursaphelenchus mucronatus [Caenorhabditis elegans. As noted in We selected all published parasitic nematode whole ESP catalogues published before May 2020 and produced experimentally by MS  using anontortus ,37, Ancy caninum ,39, Helis bakeri ,40, Nippiliensis , Necatorericanus , Strongyes ratti , Strongyuelensis , Ascarisris suum –47, Diro immitis ,49, Bruga malayi –52, Onch ochengi , Litomosmodontis , Spirocerca lupi , Gnathosinigerum , Trichuris muris , and Triris suis . Plant pncognita , Bursaphlophilus ,60, and cronatus . As a noComparative secretome analysis among ecologically diverse species may help prioritize proteins that play essential roles in parasitism for functional studies and may contribute to our understanding of the molecular basis of host-parasite specificity. To do so, protein families and orthology analyses allowed us to re-analyze all collected data simultaneously with the same tool, and resulted in a sequence-based clustering, independent of initial annotation.B. mucronatus and B. xylophilus in both analyses, as these datasets comprised mixed stages.Description of the proteins based on the Pfam families and domains was performed using Pfam_scan.pl, a Perl script calling HMMER v3 to search a FASTA file against a library of Pfam HMMs . The gatA. caninum (n = 405), A. suum (n = 277), B. malayi (n = 646), B. mucronatus (n = 326), B. xylophilus (n = 1996), C. elegans (n = 184), D. immitis (n = 180), H. contortus (n = 653), H. polygyrus (n = 381), L. sigmodontis (n = 297), N. americanus (n = 198), N. brasiliensis (n = 162), O. ochengi (n = 94), S. lupi (n = 128), S. ratti (n = 220), S. venezuelensis (n = 154), T. muris (n = 145), T. suis (n = 342). The workflow comprises a DIAMOND BLAST [B. mucronatus and B. xylophilus were included in both analyses, as these datasets comprised mixed stages. Similarly, the program was run with pooled data from all available larval stages  for each species: A. suum (n = 120), B. malayi (n = 27), B. mucronatus (n = 326), B. xylophilus (n = 1996), G. spinigerum (n = 21), H. contortus (n = 834), L. sigmodontis (n = 20), M. incognita (n = 472), N. brasiliensis (n = 31), S. lupi (n = 248), S. ratti (n = 334), S. venezuelensis (n = 415), T. suis (n = 94). Resulting data were deposited in Mendeley Data .Protein sequences from each dataset were aligned against each other  using OrthoFinder –67 with ND BLAST  all-versND BLAST ) to finaProtein sequences were aligned using MAFFT . IdenticC. elegans ESP led to the identification of 184 proteins. C. elegans ESP were predicted to have a signal peptide, with 171 proteins presenting a putative cleavage site.MS analysis of N. americanus. Stratification by species (upon pooling datasets for the same species) identified 5 Pfam accessions common to all parasitic species examined but absent in C. elegans. Two of them were enolase (PF03952.16 and PF00113.22); the other 3 were fructose-bisphosphate aldolase (PF00274.19), C-terminal domain of 1-Cys peroxiredoxin (PF10417.9), and alkyl hydroperoxide reductase (AhpC)/thiol specific antioxidant (TSA) family (PF00578.21). Thirty-nine Pfam accessions were unique to the plant parasites , but no Pfam was specifically associated with animal parasites. No Pfam was found to be clade-specific, although 2 accessions were unique to T. muris and T. suis : C8 (PF08742.11) and calponin homology (PF18383.1) domains. Similarly, no pattern was discernable with host localization (GI tract vs other tissues). Detailed Pfam accessions are provided in Representatives of the transthyretin-like protein family (PF01060.23) were found in ESP from adult worms of all 18 species included in this analysis, while cyclophilin-type peptidyl-prolyl cis-trans isomerase (PF00160.21) was present in all adult secretomes except G. spinigerum : aconitase (PF00330.20), aconitase C-terminal domain (PF00694.19), and lipocalin (PF00061.23). Apyrase (PF06079.11) was common to larvae of the two clade V parasites examined (H. contortus and N. brasiliensis).Clustering by species yielded 13 datasets for larvae  for comparison. No Pfam accession was common to all. Cyclophilin-type peptidyl-prolyl cis-trans isomerase (PF00160.21) was found in larval secretions from all species except erum see . Forty-sB. malayi  was the species with the fewest assigned genes (66.3%); ≥ 79% of ESP were assigned to orthogroups for all other species. A fraction of proteins could not be assigned to orthogroups, due to i) protein sampling  and ii) varying and relatively small dataset sizes. Maximum likelihood phylogenies of 10 highly represented orthogroups of interest  and comparison with the species tree based on the ParaSite database [10.17632/2skvdrf3rt.1; https://data.mendeley.com/datasets/2skvdrf3rt/1).Using adult ESP, OrthoFinder assigned 5783 genes  to 905 orthogroups. An orthogroup is a group of genes descended from a single gene in the last common ancestor of a group of species and is identified from a blast all-versus-all analysis ,65. B. mC. elegans. Peptidyl-prolyl cis-trans isomerase was found in all datasets except N. americanus, whereas fatty-acid and retinol-binding protein 1 was missing in both Trichuris spp. (clade I species) only. As many as 19 orthogroups appeared unique to Trichuris spp. (clade I), and only one was found to be unique to the four clade IV species . No orthogroup was unique and specific to clade III or V nematodes. Plant parasites (Bursaphelenchus spp.) shared 100 unique orthogroups. Localization in the host did not reveal further patterns. Orthogroups containing 14-3-3 protein (OG0000034), peroxiredoxin/AhpC/TSA family (OG0000027), and galectin (OG0000004), were detected in 16/18 species. Nucleoside diphosphate kinase (OG0000064), actin (OG0000022), malate dehydrogenase (OG0000041), triosephospate isomerase (OG0000044), glutathione S-transferase (OG0000009), protein disulfide isomerase (OG0000012), and glyceraldehyde 3-phosphate dehydrogenase (OG0000053) were detected in 15/18 species.The orthogroup corresponding to transthyretin-like proteins was found consistently in all adult ESP  and 105 (B. xylophilus) species-specific orthogroups were detected in all species except B. mucronatus, for which no orthogroup was found to be unique to this species.Between 1  are shown in Figs Present in all secretomes except of the nonparasitic Nematode parasite ESP are distinct from the set of somatic proteins, in class and/or abundance . Many ofin silico [in vitro cultivation used to obtain the preparations analyzed in this study varied as well. Culture conditions are expected not to reflect accurately the in vivo situation. Similarly, maintenance of C. elegans on agar plates is an artificial situation; standardized E. coli cultures do not belong in the nematode’s diet in the wild [C. elegans ESP contribute heavily to defense mechanisms against Gram-negative bacteria (such as E. coli) might be explained by culture conditions. A further limitation of this comparative work was only one secretome from a non-parasitic species fulfilled our selection criteria. Hence, the current analysis of traits of parasitism is preliminary and will require validation with additional species. Nonetheless, our work provides a functional dimension  to these ESP catalogs, and common trends and cluster-specific differences have emerged.This work presents the first comprehensive comparison of the protein components of nematode ESP from 20 species  based on experimentally-generated samples. Our results are in overall qualitative agreement with a similar study, based on 31 nematode secretomes predicted n silico . In thisthe wild . Thus, lOne reason for performing this analysis is that the complex composition of parasite ESP (hundreds of proteins in some cases) confounds the ability to identify those related to virulence and hence the success of an infection. Comparative analysis between ecologically diverse species may help prioritize proteins that play essential roles in parasitism for functional studies and may contribute to our understanding of the molecular basis of host-parasite specificity. The orthology analysis allowed to re-analyze all collected data simultaneously with the same tool, and resulted in a sequence-based clustering, independently of their initial annotation, reported in individual papers.M. incognita [A number of classical protein signatures among parasitic nematode secretomes were previously identified. These include cytokine homologues, C-type lectins and galectins, protease inhibitors, antioxidants, and venom allergen homologues . In addincognita .C. elegans, as this highly studied, free-living nematode has been a powerful model in comparative genomics and proteomics studies [C. elegans, which for instance, was not found to secrete aldolase or enolase, peroxiredoxin/AhpC/TSA family, or galectin.We included the model organism  studies . Compara studies . Indeed,N. americanus ESP. In vertebrates, transthyretin-related proteins are transporters of thyroid hormones and retinoids, while putative functions vary in invertebrates [M. incognita to regulate host plant cell growth, contribute to establishment of infection and participate in immune evasion [Ostertagia ostertagi, however, secondary structure predictions do not support the ability to achieve the conformation of transthyretin-like proteins necessary to transport lipids or thyroid hormones, which was confirmed in assays using recombinant protein [C. elegans, as transthyretin-like proteins, which show characteristics similar to neuropeptides [de novo synthesis. Fatty acid and retinol binding (FAR) proteins were common across the adult secretomes of 16/18 species reviewed here. These highly conserved proteins may play a critical role in the sequestration of a broad range of essential fatty acids, retinoids and other nutrients for delivery from the host to the parasite [Transthyretin-like proteins were identified in all adult secretions by both Pfam and orthology analyses, while cyclophilin-type peptidyl-prolyl cis-trans isomerases were absent only from tebrates . Transth evasion . In the  protein . Neuronapeptides . Nematodparasite . Many paparasite . Hydrophparasite . Nematodparasite .C. elegans ESP, several protein families may be specifically associated with parasitism, in agreement with the orthology analysis; they include the glycolytic enzymes enolase and aldolase, a peroxiredoxin, and an alkyl hydroperoxide reductase (AhpC) and thiol specific antioxidant (TSA). This is in line with recent data on the contents of C. elegans extracellular vesicles (EVs), in which these proteins were also absent [H. contortus, GAPDH has also been shown to bind to and inhibit the activity of complement C1q and C3, suggesting a role in host immunomodulation [C. elegans, while GAPDH is present. To conclude whether the secretion of glycolytic enzymes reflects an adaptation to a parasitic lifestyle will require further evidence based on secretome analysis from additional non-parasitic species. At this stage, we cannot exclude the possibility that the absence of these ESP represents a C. elegans-specific trait. In addition, whether these enzymes are active in the extracellular milieu remains to be determined.Absent from o absent , while eo absent . Togetheo absent ,87. Addio absent ,89. For o absent . In T ceo absent ,91. In Hdulation ,93. Plandulation ,94. In hdulation ,96. MBP-dulation ,97. It iGene duplication is a major driver of functional diversification of proteins. This is particularly relevant in the context of parasitism, where an evolutionary arms race between host and parasite is in place, and with events such as host range expansion. In parasitic nematodes, it is likely that rapid gene family expansions occur frequently, along with switches in secretory capacity, to facilitate adaptation to specific niches . ConsistGlycolytic enzymes lacking signal peptides, the mechanisms by which these moelcules are exported to the extracellular space are not completely resolved. However, they have been detected in EVs of various origins, also from helminths .C. elegans, B. malayi, and S. venezuelensis adults in this comparative analysis, glutathione S-transferase (GST) was described  for the free-living nematode in response to oxidative stress in general and nutrient deprivation [O. volvulus [B. malayi secretome as well, a GST had been reported, which has however been assigned to another orthogroup in the present analysis; at the time of writing, this sequence was no longer annotated as a GST, but may have GST-like activity. GST-like proteins appear to fulfil other functions, including immunomodulation. OvGST2 may protect the parasite from host-mediated lipid peroxidation, while OvGST1 was shown to generate eicosanoids, the anti-inflammatory properties of which may influence dendritic cell migration or chemotaxis of different cell types [D. immitis, A. ceylanicum, and H. contortus [Reactive oxygen species (ROS) and redox signals functionally regulate various aspects of host-pathogen interactions, from pathogen entry through protein redox switches and redox modification, to phagocyte ROS production and control of phagolysosome function (and hence antigen processing) . Parasitrivation ,101, andvolvulus . In a B.ll types ,103,104.ll types . Peroxirontortus –108.Protein disulfide isomerase (in 15/18 secretomes) belongs to a redox chaperone family with implications in many cellular processes. It participates in the maintenance of stable protein conformations. Host-derived protein disulfide isomerase determines the stability of antigen-MHC class I complexes and their transport to the plasma membrane, inhibits protozoan parasite uptake by immune cells via redox mechanisms, and modulates their translocation to phagosomes and ROS production by NADPH oxidase .N. amercicanus, function in extracellular signaling. For instance, some secreted cyclophilins induce chemotaxis or integrin-mediated immune cell adhesion [Peptidyl-prolyl cis-trans-isomerases , present in 17/18 secretomes and absent from adhesion .Mycobacterium tuberculosis to evade the innate immune system by promoting phagosome maturation arrest in macrophages and by inactivating small GTPases necessary for the production of ROS [Nucleoside diphosphate kinase, in addition to functions in the maintenance of intracellular nucleotide pools, serves as a mechanism in n of ROS .Over 40% of ESP released by plant parasites were ribosomal proteins. If their extracellular function(s) is/are unclear to date, targeting them by RNAi often results in a significant decrease in worm viability .C. elegans secretome . Similarly, we identified two glycolytic proteins that were absent in the non-parasitic species S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Mycobacterium abscessusin vitro and in a mouse model of infection. This adds a novel advanced lead compound to the M. abscessus drug pipeline and supports a strategy of screening chemical matter generated in TB drug discovery efforts to fast-track the discovery of novel antibiotics against M. abscessus.Cyclohexyl-griselimycin is a preclinical candidate for use against tuberculosis (TB). Here, we show that this oral cyclodepsipeptide is also active against the intrinsically drug-resistant nontuberculous mycobacterium  Mycobacterium abscessus causes difficult-to-cure lung disease. Multidrug regimens are administered for months to years and typically contain an oral macrolide (azithromycin or clarithromycin) and intravenously administered amikacin, imipenem/cefoxitin, or tigecycline. However, cure rates are low (<50%), and patients often undergo surgical lung resection, if feasible (–M. abscessus drug pipeline is thinly populated and largely focused on repurposing and reformulation of approved antibiotics. De novo drug discovery efforts (new chemotypes and/or new targets) are hindered by extremely low hit rates in screens attempting to identify chemical starting points  antibiotics, including all first-line drugs . The mechanism of action of many anti-TB leads has been elucidated, and pharmacokinetic (PK) properties have been optimized to enable proof-of-concept studies in animal models. Prioritization of advanced TB leads avoids the high attrition encountered in early lead optimization due to failure to introduce favorable PK properties and thus should accelerate the drug discovery process for M. abscessus. To leverage these advances, we screened TB leads against M. abscessus and identified several novel anti-M. abscessus compounds with demonstrated in vivo activity, including inhibitors of ATP synthase  hub. The protein surrounds double-stranded DNA and functions to recruit a diverse range of accessory proteins involved in DNA metabolism –18. DnaN–nockdown . Fluores as the readout for growth . Interestingly, the bactericidal activity of CGM against M. abscessus K21 was lower than that against M. abscessus ATCC 19977. Despite showing similar MICs against both strains (∼0.5 μM)  , higher cultures . The reacultures , treatmein vivo efficacy of CGM, we infected 8-week-old female NOD.CB17-Prkdcscid/NCrCrl (NOD SCID) mice (Charles River Laboratories) by intranasal delivery of 106 CFU M. abscessus K21 as described previously (M. abscessus infection.To assess the eviously . In thiseviously . Drugs oeviously . Compareeviously . CFU redeviously . Thus, CM. abscessus complex in vitro. The advanced anti-TB lead compound displayed bactericidal activity in vitro and reduced the bacterial lung burden in a mouse model of M. abscessus infection. This work adds a new advanced lead compound to the preclinical M. abscessus drug discovery pipeline and suggests that the new anti-TB drug candidate could be explored for the treatment of M. abscessus lung disease. The demonstration that yet another TB active displays anti-M. abscessus activity supports the paradigm of exploiting chemical matter generated for TB drug discovery to accelerate de novo drug discovery for M. abscessus.In conclusion, we show that the cyclohexyl analog of griselimycin, CGM, is broadly active against the


Meloidogyne incognita (Kofoid & White) Chitwood), reniform (Rotylenchulus reniformis Lindford & Oliveira), and lesion nematodes (Pratylenchus penetrans (Cobb) Filipjev & Schuurmans Stekhoven) are plant-parasitic nematodes that feed on soybean (Glycine max (L.) Merr.) roots, limiting seed production. The availability of resistance in soybeans to these nematodes is limited. However, new sources of resistance can be discovered in wild relatives of agronomic crops. Perennial Glycine species, wild relatives to soybean, are a source of valuable genetic resources with the potential to improve disease resistance in soybean. To determine if these perennials have resistance against nematodes, 18 accessions of 10 perennial Glycine species were evaluated for their response to M. incognita and R. reniformis, and eight accessions of six perennial Glycine species were evaluated for their response to P. penetrans. Pot experiments were conducted for M. incognita and R. reniformis in a growth chamber and in vitro experiments were conducted for P. penetrans. We found both shared and distinct interactions along the resistance-susceptible continuum in response to the three plant-parasitic nematode species. Ten and 15 accessions were classified as resistant to M. incognita based on eggs per gram of root and gall index, respectively. Among them, G. tomentella plant introductions (PIs) 446983 and 339655 had a significantly lower gall index than the resistant soybean check cv. Forrest. Of three R. reniformis resistant accessions identified in this study, G. tomentella PI 441001 showed significantly greater resistance to R. reniformis than the resistant check cv. Forrest based on nematodes per gram of root. In contrast, no resistance to P. penetrans was recorded in any perennial Glycine species.Root-knot ( Meloidogyne incognita (Kofoid & White) Chitwood), reniform nematode (Rotylenchulus reniformis Linford & Oliveira), and lesion nematode (Pratylenchus penetrans (Cobb) Filipjev & Shuurmans Stekhoven) are common plant-parasitic nematodes that infect soybean (Glycine max (L.) Merr.) and other crops, causing yield losses   88788 is the most commonly used source of resistance in soybean against the soybean cyst nematode  . Howeverniformis . Among 7ectively .Meloidogyne spp. and R. reniformis and resistant soybean lines have been identified  originated from wild tomato relative Solanum peruvianum (S. lycopersicum L.)  and G. tomentella PI 505214 were chosen because of the availability of sequence information  . Wild pe Glycine . Perennie to SCN . We hypoeviously ; G. latiormation .Glycine species and five soybean cultivars were obtained from the USDA-ARS Soybean Germplasm Collection (https://www.ars-grin.gov/)  and two G. tomentella accessions, PIs 446983 (Papua New Guinea) and 339655 . Chromosome numbers varied from 40 to 80 .Eighteen perennial in.gov/) . All accGlycine seeds were scarified with a razor blade by slightly cutting the seed coat on the opposite side of the hilum. Seeds were germinated for 5 to 7 days on wet tissue paper in a plastic box for M. incognita tests and on Sun Gro® Sunshine® LC1 Grower Mix  in petri dishes for R. reniformis tests. Seedlings were planted in steam pasteurized torpedo sand, for M. incognita, or sandy loam , for R. reniformis, in SC10 Conetainers . Three weeks after planting, the seedlings were inoculated with nematodes. Seeds of susceptible  and resistant soybean checks (cv. Forrest) were germinated following the same methods above (but not scarified) and planted in torpedo sand or sandy loam 1 week prior to inoculation.Perennial P. penetrans, root explants were prepared on agar media. Scarified perennial Glycine seeds were surface disinfected in 0.5% sodium hypochlorite (NaOCl) for 5 min and rinsed three times with sterilized distilled water. Susceptible check cv. Lee 68 and other soybeans, PI 88788 and cv. Williams 82 were surface disinfected in 0.5% NaOCl for 20 min and rinsed three times with sterilized distilled water. Five seeds of each accession were transferred onto a Murashige and Skoog (MS) (For oog (MS)  solid meM. incognita, originally isolated from soybean in southern Illinois (generous gift from Jason Bond) and identified using polymerase chain reaction (PCR) with species-specific primers (S. lycopersicum) cv. Tiny Tim in the greenhouse. Tomato roots with root galls were cut into small pieces and mixed with 200 ml of 0.5% NaOCl and vigorously shaken manually for 4 min to release eggs from the gelatinous matrix  in College Station, Texas, was maintained on soybean cv. Macon or cv. Braxton in the greenhouse (generous gift from Martin Wubben). To extract vermiform R. reniformis, the roots were removed, soil was suspended in water, and poured through 841- and 38-μm sieves  and identified based on morphological characteristics and mitochondrial cytochrome c oxidase subunit 1 and 28S rDNA sequences  weekly after transplanting. Two soybean genotypes for susceptible and resistant checks were included in each experiment  with five replications and each test was repeated once. Data were not collected from a few experimental units where seeds did not germinate or where seedlings were too small to inoculate. periment . The susperiment . R. reniout host .P. penetrans tests were conducted in a growth chamber at 25°C and 16 hr of fluorescent light per day for six days. G. max cv. Lee 68 was included as a susceptible check  8 weeks after inoculation  per gram of fresh root 10 weeks after inoculation. To extract R. reniformis, soil was washed from the plant root, suspended in water, and poured through 250- and 38-μm sieves. Nematodes collected on the 38-μm sieve were further processed by sucrose-centrifugation (R. reniformis eggs were extracted from the roots as described above after measuring the fresh weight of the washed plant roots.The response to fugation . R. reniP. penetrans was based on nematode counts in the roots following acid fuchsin staining (The response to staining . SeedlinM. incognita eggs per gram of root data and P. penetrans nematodes per gram of root were log (x + 1) transformed and R. reniformis nematodes per gram of root data were log (x) transformed to meet normality and homogeneity of variance assumptions. The ANOVA analyses were done with JMP Pro 14.2.0 Fit X by Y or Fit Model platforms. Mean separations were done using JMP Pro 14.2.0 Tukey-Kramer HSD test at α = 0.05 . The analysis of variance (ANOVA) was done for each trial individually and for both tests pooled if homogeneity of variance was not significant between trials. α = 0.05 .Glycine species were categorized as susceptible (S), moderately resistant (MR), and resistant (R) based on the statistical comparison with susceptible and resistant soybean checks: S ≥ susceptible check; susceptible check > MR > resistant check; R ≤ resistant check. Ratings were determined by combining trial data for both M. incognita and R. reniformis, while P. penetrans trial data were kept separate.Nematode resistance levels in perennial P > 0.05) between trials, so data were pooled for analysis  had significantly fewer eggs per gram of root than the susceptible check cv. Pickett 71, and fewer root galls  between trials so data were pooled for analysis  were significantly more susceptible to R. reniformis than the susceptible check PI 88788. Only G. tomentella PI 441001 had a significantly lower number of R. reniformis per gram of root than cv. Forrest.The Bartlett test for homogeneity of variance was not significant  between trials so data were analyzed separately  enabled hybridization between G. max cv. Dwight (2n = 40) and G. tomentella PI 441001 (2n = 78)  . Hybrid o Dwight . StudiesHG types . Our stuGlycine species for their response to M. incognita and R. reniformis, and eight PIs for response to P. penetrans. PIs were selected based on prior evaluation confirming a resistant or susceptible reaction to SCN   . PI 4409hyrhizi)  and suscchyrhizi . PI 4469M. incognita based on the gall index, but susceptible based on egg production (eggs per gram of root). This group included G. argyrea PI 509451, G. curvata PI 505167, G. microphylla PI 505188, PI 509487, G. tomentella PI 505214, and PI 505238. The contrast between reproduction and gall indices in these accessions to M. incognita was also previously seen in soybean  found that, in contrast to conserved low-copy genes, gene families associated with disease resistance had undergone rapid diversification, such as genomic duplications and losses, and suggested that the rapid diversification of disease resistance genes might have been driven by pathogen-mediated pressure  using wild soybean (G. soja) identified a novel SCN-resistance locus on chromosome 19 (G. max cv. Dwight and G. tomentella PI 441001 (Glycine to soybean using CRISPR-Cas9 gene-editing technologies (Glycine species and to discover and characterize additional resistance genes, molecular and genomic studies may provide the tools needed to further develop soybean resistance to M. incognita, R. reniformis, and P. penetrans. PIs identified in this study will serve as resources in ongoing efforts to identify novel nematode resistance genes for M. incognita and R. reniformis.Perennial osome 19 . Beyond I 441001 , it may nologies . To incr


Hymenoptera comprise extremely diverse insect species with extensive variation in their life histories. The Dryinidae, a family of solitary wasps of Hymenoptera, have evolved innovations that allow them to hunt using venom and a pair of chelae developed from the fore legs that can grasp prey. Dryinidae larvae are also parasitoids of Auchenorrhyncha, a group including common pests such as planthoppers and leafhoppers. Both of these traits make them effective and valuable for pest control, but little is yet known about the genetic basis of its dual adaptation to parasitism and predation.Gonatopus flavifemur, which at 636.5 Mb is larger than most hymenopterans. The expansion of transposable elements, especially DNA transposons, is a major contributor to the genome size enlargement. Our genome-wide screens reveal a number of positively selected genes and rapidly evolving proteins involved in energy production and motor activity, which may contribute to the predatory adaptation of dryinid wasp. We further show that three female-biased, reproductive-associated yellow genes, in response to the prey feeding behavior, are significantly elevated in adult females, which may facilitate the egg production. Venom is a powerful weapon for dryinid wasp during parasitism and predation. We therefore analyze the transcriptomes of venom glands and describe specific expansions in venom Idgf-like genes and neprilysin-like genes. Furthermore, we find the LWS2-opsin gene is exclusively expressed in male G. flavifemur, which may contribute to partner searching and mating.We sequenced and assembled a high-quality genome of the dryinid wasp G. flavifemur, and present genomic resources for future in-depth comparative analyses of hymenopterans that may benefit pest control.Our results provide new insights into the genome evolution, predatory adaptation, venom evolution, and sex-biased genes in The online version contains supplementary material available at 10.1186/s12915-021-01081-6. Hymenoptera are an extremely diverse insect order with a variety of life history traits, including phytophagy, parasitism, predation, pollination, and eusociality, providing an ideal model for studying the evolutionary origin and transition of some key traits , 2. DryiGonatopus flavifemur is a common parasitoid of the notorious rice pest, the brown planthopper Nilaparvata lugens [G. flavifemur, representing the first genome sequence of the family Dryinidae. This 636.5 Mb genome assembly is much larger than most hymenopterans, due to the massive expansion of transposable element sequences. Analysis of the G. flavifemur genome highlights several positively selected genes and rapidly evolving proteins likely involved in major aspects of predatory adaptation. Gene expression changes in female adults after feeding on the preys are featured. Due to the importance of venom to the wasps, we also provide insights into the venom-associated genes, and describe specific expansions in venom Idgf-like genes and neprilysin-like genes. Finally, we identify many sex-biased genes which may be related to sexual dimorphism. In sum, our findings provide insights into the genome size evolution, parasitoid-predatory adaptation, venom evolution, and sexual dimorphism. In addition, this genome underpins further research of G. flavifemur and greatly facilitates future analyses of the trait evolution in Hymenoptera.Dryinidae provides a promising model to study the origin of predatory behavior and sexual dimorphism. a lugens –8. Its ua lugens , 10. HerG. flavifemur estimated by flow cytometry and K-mer analysis were about 601.4 and 603.4 Mb, respectively  [G. flavifemur, which is 2.8 times the length of A. mellifera (255 Mb) and 1.9 times the length of P. puparum (338 Mb). Moreover, the haploid chromosome number of Gonatopus wasps is four [G. flavifemur is 159.1 Mb, which is much larger than the maximum contig length of our assembly (81.2 Mb). Thus, the Nanopore long-read sequencing and assembly strategy, the large genome size, and low chromosome number explain the very long contigs obtained in our assembly.We generated Nanopore long reads (107X genome coverage) and Illumina short reads (58X genome coverage) from 50 male pupae for genome assembly  , 27. Suc is four . Based oNasonia vitripennis, our annotation is similar, but slightly larger than other hymenopterans. Moreover, we further identified several gene families that are important and popular in insect studies, including 66 cytochrome P450s, 17 glutathione S-transferases (GSTs), 35 ATP-binding cassette transporters (ABCs), 10 gustatory receptors (GRs), 20 ionotropic receptors (IRs), 43 olfactory receptors (ORs), 8 odorant binding proteins (OBPs), 8 sensory neuron membrane proteins (SNMPs), and 6 chemosensory proteins (CSPs)  than other hymenopteran insects, which may be explained by its relatively narrow host range  and unitary living environment (rice fields).Genome assessment using Benchmarking Universal Single-Copy Orthologs (BUSCO) indicated 99.3% of the insect gene set are present and complete Fig. g. This BG. flavifemur) for phylogenomic analyses and the following comparative genomics analyses because of their high genome qualities, popularities, and evolutionary positions. Our phylogenomic analyses were based on 2992 single-copy genes, which were firstly identified by OrthoFinder from the genomes of 13 hymenopterans . The amino acid sequences of the single-copy gene set were aligned and concatenated, followed by a phylogenetic tree construction. In this analysis, dryinid wasp G. flavifemur (from the superfamily Chrysidoidea) was placed as the sister group to all other Aculeate members  after their common ancestor diverged from the infraorder Parasitoida  [Belonocnema treatae (1.5 Gb) (NCBI RefSeq assembly accession: GCF_010883055.1). The genome size of dryinid wasp G. flavifemur is 636.5 Mb, representing a relatively large genome in Hymenoptera. The expansion of repetitive sequences  is one of the most important factors to enlarge the genome size, and this phenomenon has been reported in many insects [G. flavifemur genome contains massive repeat sequences, comprising approximately 60.7% of the whole genome. TEs account for around 59.9% (381.7 Mb) of the G. flavifemur genome, with DNA transposons being the most abundant TE group  , while gene region length shows weak correlation with genome size . This result suggested that TE is a strong factor to drive genome size enlargement in the evolution of Hymenoptera. This pattern is supported by a previous study focused on aculeates [Athalia rosae (155.8 Mb) has a moderate genome size and a low TE content (4.6%), which may imply the ancestral state of hymenopteran genome. During the evolution of Hymenoptera, TE expansion likely happened independently after species divergence, resulting in genome size enlargement. In addition to the TE expansion of G. flavifemur, we found another obvious example, P. puparum, which also has abundant expanded TEs (about 40.1%) in its genome [2 = 0.77, p = 8.0e−05, Pearson’s test corrected by the phylogeny). We also detected moderate positive correlations between the content of long terminal repeat retrotransposons (LTRs) , long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) , miniature inverted-repeat transposable elements (MITEs) , and the genome size. This result suggested that the expansions of other TEs  also contribute to genome size evolution in Hymenoptera.The hymenopteran genomes are moderate in size (80% are between 180 and 340 Mb) based on current sequencing projects . However(3.3 Gb)  and the  insects –34. Unsup 40.3%; 56.3 Mb  , hAT , Mutator , and Helitron  , with a small genome (140.1 Mb) and low TE content  are present in all hymenopteran insects analyzed in this study. The G. flavifemur genome contains 727 Aculeate-specific genes, which is more than other aculeates. GO enrichment analysis revealed that these Aculeate-specific genes are enriched in DNA integration, DNA recombination, cellular aromatic compound metabolic process, and nitrogen compound metabolic process  using OrthoFinder among G. flavifemur-specific evolutionary traits, including the origin of predation, we performed genome-wide selection analysis and gene family evolutionary analysis to screen for gene changes that occurred in the G. flavifemur genome.Unlike most of the parasitoid wasps in the infraorder Parasitoida , the dryinid wasps  are usually both parasitoids and predators on their hosts . As paraG. flavifemur in the phylogeny in Fig. p < 0.05, FDR-adjusted) to define the significance of each candidate gene. Only those genes showing positive selection signals in both methods were used for the following study. In total, our strict screening criteria identified 183 genes that have evidence of positive selection in the G. flavifemur genome . We found some positively selected genes are related to mitochondrial functions, including NDUFB3, NDUFA9, mtTFB1, Myg1, and PTCD3 [NDUFB3 and NDUFA9) are in the mitochondrial electron transport chain (Complex I), which transfers electrons from NADH to ubiquinone [G. flavifemur’s hunting. In addition, a number of positively selected genes are involved in actin cytoskeleton, muscle contraction, and motor activity, including TLN1 [TBCE [Scgβ [SPG11 [ALS2 [TWF1 [SIM [G. flavifemur. Interestingly, our positively selected gene set includes a Piwi-like AGO3 gene, which belongs to the piRNA pathway. This AGO3 protein interacts with piRNA and plays a central role during meiosis by repressing TEs and preventing their mobilization, which is essential for germline integrity [G. flavifemur genome and some TEs may still be active. Thus, we hypothesize that this selected AGO3 of G. flavifemur might have functions on repressing TEs in the germline.First, 2992 single-copy genes were used for screening the signatures of positive selection on the terminal branch of nd PTCD3 –40  based on their total branch lengths . Then, we assigned ranks to proteins (from 1 to 2992) based on their branch lengths of G. flavifemur branches . We next binned proteins into groups of 300 based on their total branch length rank, which could avoid overrepresentation among any protein categories based on general evolutionary rates. In each bin, we selected the proteins with the top 10% largest discrepancy in rank between the G. flavifemur branch length rank and the total branch length rank, as rapidly evolving proteins  , one visual system homeobox 2 protein , and one cryptochrome-1 protein (related to circadian rhythm) were identified. Together, our searches for positively selected genes and rapidly evolving proteins revealed several genes or proteins involved in energy production and motor activity, which suggests that altered genes or proteins may contribute to the adaptation of predatory behavior in G. flavifemur.Additionally, we performed a rank-based branch length comparison method to study the protein evolutionary rates of the p genome . In thisG. flavifemur genome. A total of 434 gene families were expanded in G. flavifemur comparing to the common ancestor of the Aculeate, including chitinase-like proteins (glycosyl hydrolase family 18), neprilysins , trypsins, venom carboxylesterases, esterases, G-protein-coupled receptors, and several transcription factors  genes were expanded in G. flavifemur, and they may contribute to slow-twitch fiber type specificity during myogenesis and regenerating muscles [G. flavifemur than in carcass , adult male, pupa, and larva, suggesting that they might be venom genes and play important roles in parasitoid-host interactions. See below for the detailed analyses about the chitinase/chitinase-like gene family and the neprilysin family.We next investigated the gene family expansions and contractions in the ors Fig. a. The si muscles . InteresG. flavifemur, which acts as both parasitoid and predator on its host. We hypothesize that the venom of G. flavifemur might have similar functions with the venoms of both parasitoid wasps and predatory wasps, and it is expected to play a key role in both altering host’s metabolism and immunity (like many parasitoid wasps’ venom), as well as paralyzing the host temporarily (like many predatory wasps’ venom).Among most of the hymenopteran insects, venom plays essential roles in their life. For example, parasitoid wasps use venom to manipulate the metabolism and immunity, and gene expression of the host to establish a suitable environment for wasp larvae –64, whilG. flavifemur, we analyzed the RNA-seq datasets derived from venom gland and carcass  respectively. The venom gland is a highly specialized organ, which produces the venom components. We found that there is only a small set of genes highly expressed in the venom gland. For example, only 157 and 474 genes account for 80% and 90% of expression in the venom gland of G. flavifemur; however, 979 and 2297 genes account for 80% and 90% of expression in adult female  VGGs must be among the top500 expressed genes  in the venom gland transcriptome; (2) VGGs must be significantly highly expressed in the venom gland relative to the carcass (q < 0.05); (3) VGGs must remain low expression levels in the carcass (median FPKM < 50). In total, 154 VGGs were identified in G. flavifemur  of G. flavifemur . Such a large number of venom-expressed chitinase-like genes was not reported in any other wasps, and might play an important role during the parasitism and hunting of G. flavifemur. It could occur due to the gene duplications after a single venom gene recruitment, or many independent venom gene recruitment events after gene duplications, or a complex evolutionary history that includes both cases above. Additionally, the venom gene recruitment of chitinase-like genes might be due to the lateral gene transfer (LGT), and it has been reported in some parasitoids in Chalcidoidea [Notably, a number of chitinase-like genes  were found to be specifically highly expressed in the venom gland of cidoidea . Howeverp < 0.05, FDR-adjusted; Additional file G. flavifemur might regulate the host’s cuticle development and prevent the host from dying due to excessive hemolymph loss. Parasitized hosts will be alive until the mature wasp larvae leave the hosts for cocooning and pupation [G. flavifemur venom might be involved in the wound healing process to ensure the host is alive.Because of the abundant chitinase-like genes and clotting-related proteases (8 genes) in the venom gland, the GO enrichment analysis showed that the VGGs are enriched in carbohydrate metabolic process, imaginal disc development, coagulation, wound healing, proteolysis, cuticle development  in the VGGs, and 7 of them contain predicted signal peptides , 1 lepidoptera genome (Bombyx mori), 1 coleopteran genome (Tribolium castaneum), 1 diptera genome (Drosophila melanogaster), and 1 hemipteran genome (Acyrthosiphon pisum). The four non-hymenopteran genomes we analyzed here allow us to determine the ancestral state of chitinase-like gene family of Hymenoptera. In total, 260 GH18 domain-containing proteins were found and 61 of them were in the G. flavifemur genome, representing the most abundant one when compared to the other 16 genomes. Maximum likelihood phylogenetic analysis indicated that the chitinase-like genes in group 4 (7 genes) and group 5 (47 genes) were expanded in the G. flavifemur genome, whereas the gene counts in other chitinase groups among 17 insect genomes were highly conserved  in T. castaneum and parasitoid wasp Microplitis mediator and Cotesia chilonis show that although IDGFs retain the glutamate residue in the active site motif, a D to A substitution in the same motif (from DxxDxDxE to DxxDxAxE) also results in the loss of function of IDGFs  in the G. flavifemur genome, indicating the tandem duplications of Idgf genes. The largest tandem array contains 6 Idgf genes  while the last one is G. flavifemur-specific. Moreover, this G. flavifemur-specific Idgf displayed a strong venom-gland biased expression pattern. Among 47 Idgf genes, 28 were assigned as VGGs in our analysis , which are a small family of chitinase-related secretory proteins found in many insects –87. Theyinanitus , 90. TheIdgf and group 4 chitinase genes with venom-gland high expressions might have important functions in G. flavifemur-host interaction. Further studies are needed to investigate the function of these venom chitinase-like genes.An additional gene from the group 4 chitinase subfamily was also highly expressed in the venom gland and very lowly expressed across development , which is much more abundant than any other insects in this study, ranging from 5 to 42 . In Parasitoida, venom neprilysin-like genes were found in a chalcid wasp Pachycrepoideus vindemmiae, and 3 figitid wasps Leptopilina boulardi, L. heterotoma, and Ganaspis sp., 4 braconid wasps M. mediator, M. demolitor, Microctonus hyperodae, Aphidius ervi, and an ichneumonid wasp Hyposoter didymator. In Aculeate, we found additional venom neprilysin-like genes in a bethylid wasp Scleroderma guani, a solitary hunting wasp Eumenes decorates and an emerald cockroach wasp Ampulex compressa. We then summarized the presence or absence of the venom neprilysin-like genes in total 31 hymenopterans and mapped the information to their phylogeny. Our results show that the recruitments of venom neprilysin-like genes are mainly scattered throughout the Hymenoptera phylogeny. This indicates that most recruitment events of venom neprilysin-like gene occurred independently during the radiation of Hymenoptera .Venom neprilysins have been reported in many venomous animals, such as jellyfishes, snakes, and centipedes and spiders, suggesting many convergent venom recruitments , 78, 80.G. flavifemur female adults may be beneficial to reproduction. To test our hypothesis, we analyzed the transcriptome data derived from the prey-feeding females (protein-rich diet) and sucrose-feeding females to characterize differential expressed genes between these two treatments. In total, 24 genes were significantly upregulated in the prey-feeding females when compared to the sucrose-feeding females  after feeding on the preys  were enriched in nucleosome assembly, meiotic cell cycle process  showed a striking enrichment of GO terms associated with sensory perception, detection of stimulus, and cell-cell signaling  opsin genes, while the rest species have two LWS-opsin copies , while the LWS2-opsin of P. puparum was only 2.89 higher expressed in the males (q = 2.24e−06). In addition, the LWS2-opsin of N. vitripennis did not show a male-biased expression pattern , providing a valuable resource to facilitate our understanding of Hymenoptera evolution. In addition, G. flavifemur has many unique biological characteristics, including parasitism, predation, and sexual dimorphism.Herein, by combining both Nanopore long-read sequencing and Illumina short-read sequencing strategies, we generated a high-quality reference genome of the pincer wasp G. flavifemur. These findings expand our understanding of the hunting behavior evolution in Hymenoptera. By incorporating transcriptomic data, we found that 3 yellow genes  were significantly activated in female G. flavifemur after feeding on the preys. It has been reported that some members of the yellow gene family are associated with reproductive maturation. For example, yellow-g and yellow-g2 play a female-specific role in the egg development of Drosophila and Aedes [yellow genes of silkworm Bombyx mori have a high transcription level in both ovary and testis, suggesting their reproduction-related functions [G. flavifemur.In this study, our comparative genomics analysis highlighted a number of positively selected genes and rapidly evolving proteins involved in energy production and motor activity, which may play roles in the predatory adaptation of nd Aedes , 96. Morunctions . TherefoIdgf genes and neprilysin-like genes. The functions of Idgf genes in insects are diverse, and they have been proved to participate in cuticle formation, wing development, larval and adult molting, immune response, antimicrobial response, and hemolymph clotting [G. flavifemur might play various potential roles, for example, regulating the development of the hosts, participating in the wound healing of the hosts, and immune-related functions against the hosts and bacteria. On the other hand, neprilysin specializes in the role of metabolizing and regulating molecules in the mammalian nervous systems; for example, it can inactivate peptide transmitters and their modulators [G. flavifemur venom might have a powerful paralyzing effect on the host, which is of significant benefit to hunting and parasitism. Indeed, the temporary paralyzing effect of dryinid wasp’s venom has been reported before [Venoms are key evolutionary innovations in most hymenopteran insects and have been used for predatory, defensive, and reproductive purposes. Our comprehensive study on the venom gland identified significant expansions and unique expression patterns of clotting , 99, 100clotting . Here, wdulators , 102. Thd before , 103, anG. flavifemur is much larger than most hymenopterans. The expansion of TEs, especially from DNA transposons, is a major contributor to the genome size enlargement in G. flavifemur. TE expansions and insertions can cause a variety of changes in the host genome, such as chromosomal rearrangements, gene disruptions, and gene expression regulations, some of which may be of benefit to adaptation [G. flavifemur. We envision further investigations on how TE expansion benefits the adaptation of G. flavifemur.The genome size of aptation , 104–107aptation , 109. Inaptation . We therG. flavifemur to understand the genetic basis of these key innovations. Compared to other hymenopterans, our findings highlight that the TEs, especially DNA transposons, have massively expanded in the G. flavifemur genome, resulting in the genome size enlargement. Our genome-wide screens locate a number of positively selected genes and rapidly evolving proteins involved in energy production and motor activity, which may contribute to the predatory adaptation of G. flavifemur. We also show that 3 female-biased, reproductive-associated yellow genes in adult females expressed significantly higher after feeding on the preys, which may be beneficial to the egg production. This may explain the advantage of their unique predatory behavior. In addition, our transcriptomic analyses and following gene family analyses reveal the unique venom characters of G. flavifemur, such as the expansions of venom Idgf-like genes and neprilysin-like genes. Furthermore, we identify sex-biased genes based on the differences of gene expression between adult females and males, and observe an extremely male-biased LWS2-opsin gene in G. flavifemur. These results advance our understanding of the genome architecture, predatory adaptation, venom gene evolution, and sex-biased genes in G. flavifemur, and stimulate further comparative analyses of hymenopterans.Parasitoid wasps in the family Dryinidae display several interesting characteristics, such as the predatory behavior of adult females and the distinct sexual dimorphism. In this study, we present the genome of the dryinid wasp G. flavifemur were provided by Dr. Qiang Fu from his laboratory. The wasp colony was originated from about 50 wasps of a field population, which was collected at Hangzhou, China, in 2018. The colony of the hosts N. lugens was first collected from rice fields at Hangzhou, China, in 2018. Both parasitoid wasps and the hosts were maintained in the laboratory. The N. lugens was reared continuously on a susceptible rice variety  under laboratory conditions at 28 ± 1 °C, 65 ± 5% relative humidity (RH), 3500 ~ 4000 Lux and a photoperiod of 16: 8 h (light: dark) [G. flavifemur were reared on 4–5th instar brown planthoppers under the same conditions. The details of samples for genome and transcriptome sequencing are summarized in Additional file The dryinid wasps t: dark) . The parG. flavifemur. High-quality genomic DNA for de novo sequencing was extracted from 50 haploid male pupae using the sodium dodecyl sulfate (SDS)-based DNA extraction method followed by purification with VAHTS DNA Clean Beads  according to the standard procedure provided by the manufacturer. The quality and concentration were then assessed by 1% agarose gel electrophoresis, NanoDrop™ One UV-Vis spectrophotometer  and Qubit® 3.0 Fluorometer . For long library preparation, qualified DNA was size-selected (> 10 kb) using the BluePippin system , then two long libraries were processed according to the Ligation Sequencing Kit  protocol and sequenced on two flow-cells using the PromethION sequencer (Oxford Nanopore). A short paired-end library with an insert size of 350 bp was constructed using a TruSeq Nano DNA HT Sample Preparation Kit (Illumina), and then sequenced on the HiSeq X Ten platform.We applied the Nanopore and Illumina HiSeq X Ten platforms to sequence the genome of 2, 20 mM 3-N-morpholinopropane sulfonic acid, 30 mM sodium citrate, and 0.1% (vol/vol) Triton X-100; pH 7.0). The homogenate was then filtered into a 1.5-mL Eppendorf tube using 38-μm nylon mesh. To remove RNAs, RNase A  was added to the homogenate  and incubated at 25 °C for 10 min. The precipitates were collected by centrifuging at 1000g for 5 min, and then suspended with 400 μL phosphate buffer (pH 7.4) and stained with 50 μg/mL propidium iodide stock solution in darkness at 4 °C for 10 min. Each sample was analyzed using the MoFlo™ XDP High Speed Cell Sorter and Analyzer  under 488-nm wavelength. Summit Software  was used to obtain the nuclei peaks. The genome size was then estimated based on the outputs, using D. melanogaster as a control  to correct Nanopore reads with -seed_cutoff = 10 k parameter. The corrected reads were then assembled into primary contigs using wtdbg2 v2.3 [We firstly used NextDenovo v1.1.1  were prepared from larva , pupa , female adult , male adult , venom gland , and carcass . In addition, to investigate the gene expression changes between prey-feeding females and none prey-feeding females, six RNA-seq libraries (insert size of 250 bp) were prepared from the female adults (5-day-old) reared on 20% sucrose water and brown planthoppers (the preys), respectively. Three biological replicates were prepared for each sample. The transcriptomes were sequenced using the Illumina HiSeq X Ten platform with paired-end libraries. Raw reads from the RNA-seq were filtered using Trimmomatic v0.38 . The cleLTRharvest [We used the Extensive de novo TE Annotator (EDTA) pipeline to construct TE libraries for each species . BrieflyRharvest  and LTR_Rharvest  for LTR Rharvest  and HeliRharvest  for DNA Rharvest ). Next, Rharvest  was usedRharvest  was thenRharvest . Tandem Rharvest . The divhttps://www.ebi.ac.uk/about/vertebrate-genomics/software/exonerate) and GenomeThreader v1.7.1 [https://github.com/TransDecoder/TransDecoder). Finally, EVidenceModeler v1.1.1 [Three approaches, as incorporated in the EVidenceModeler pipeline  , were usr v1.7.1  were user v1.1.1  was usedr v1.1.1 . We nextr v1.1.1  online sr v1.1.1  , C. chilonis (http://www.insect-genome.com/), Habrobracon hebetor , Nasonia vitripennis , P. dominula (RefSeq assembly accession: GCF_001465965.1), Solenopsis invicta (RefSeq assembly accession: GCF_000188075.2), Ooceraea biroi (RefSeq assembly accession: GCF_003672135.1), Megachile rotundata (RefSeq assembly accession: GCF_000220905.1), A. mellifera (RefSeq assembly accession: GCF_003254395.2), and A. rosae (RefSeq assembly accession: GCF_000344095.2). The basal hymenopteran A. rosae was used as an outgroup.We comprehensively considered three factors of each species for comparative genomics analyses and phylogenomic analyses, including genome quality, popularity, and evolutionary position. OrthoFinder v2.5.1  was usede et al. ), Trichoo et al. ), P. pupe et al. ), G. flaA total of 2992 one-to-one orthogroups shared by the 13 Hymenoptera species were extracted for phylogenetic analysis. The protein sequences in each orthogroup were independently aligned by MAFFT v7  and filthttps://CRAN.R-project.org/package = caper) to test the effect of the phylogeny on the correlation between genome size and TE content. We obtained a lambda parameter from 0 to 1  by fitting a linear model on the data. We then computed corrected Pearson’s correlation between the genome size and the TE content using the ape package v5.4-1 [Considering the phylogenetic relationships of the species studied, we first used the R caper package v1.0.1  was conducted to compare the null model  and the alternative model (sites under positive selection on the foreground branch). The p values were computed based on chi-square statistics with a false discovery rate (FDR), and genes with p-adjusted value less than 0.05 were identified as positive selection genes. For HyPhy analysis, the aBSREL algorithm [p values < 0.05 were considered to be under positive selection. Finally, 183 genes detected by both methods were treated as candidates that underwent positive selection.We used two packages, PAML v4.9  and HyPhlgorithm  was usedG. flavifemur branch as used in the Trichogramma genome [G. flavifemur branch length. To avoid overrepresentation among any protein category based on general evolutionary rates, we next binned proteins into groups of 300 based on the total branch length rank. Rapidly evolving proteins were defined as proteins with the top 10% largest values of the G. flavifemur branch length rank minus the total branch length rank in each group.Based on 2992 single-copy proteins, we used a rank-based branch length comparison method to identify rapidly evolving proteins on a genome . In briea genome . We thenWe used CAFE v4.2.1  to analyq < 0.05), and (c) not highly expressed in the carcass (median FPKM < 50). The signal peptide of each venom associate gene was predicted using the SignalP v5.0b [To identify the venom gland-associated genes, we firstly calculated the expression level of each transcript in the venom gland and the carcass  using RSEM v1.3.1 . To quallP v5.0b .G. flavifemur and other 12 hymenopteran genomes mentioned in the comparative genomics section using BLASTP v2.8.1 [To identify genes involved in detoxification and chemosensory, cytochrome P450s, glutathione S-transferases (GSTs), ATP-binding cassette transporters (ABCs), gustatory receptors (GRs), ionotropic receptors (IRs), olfactory receptors (ORs), odorant binding proteins (OBPs), sensory neuron membrane proteins (SNMPs), and chemosensory proteins (CSPs) protein sequences of well-annotated insects retrieved from Uniprot were used as queries to search against the predicted protein sequences from P v2.8.1  , Tribolium castaneum (RefSeq accession: GCF_000002335.3), Drosophila melanogaster (RefSeq accession: GCF_000001215.4), and Acyrthosiphon pisum (RefSeq accession: GCF_005508785.1). We next checked the putative chitinase-like genes containing the glycosyl hydrolase 18 (GH-18) domain using hidden Markov models (HMM search) [G. flavifemur based on the multiple sequence alignment generated by MAFFT v 7.471 [To annotate the GH-18 chitinase-like genes, a number of well-annotated insect chitinase protein sequences were used as queries in a BLASTP search  against the proteins of 17 insect genomes including 13 hymenopteran genomes mentioned in the comparative genomics section,  search) . Finally search)  was then search)  in IQ-TR search)  analysis search) . The con search) , 88, 155To identify neprilysin-like genes, we retrieved the well-annotated neprilysin sequences from UniProtKB/SwissProt as queries. BLASTP  was performed for protein searching in the 17 insect genomes described in chitinase-like gene section. Each protein sequence obtained was subsequently used for searching against Pfam-A  databaseA. mellifera, D. melanogaster, and T. castaneum were obtained from UniProt. They were used for searching the proteins against 13 hymenopteran genomes using BLASTP v2.8.1 [Amino acid sequences of opsin genes in P v2.8.1  . Supplementary Table 11. GO enrichment analysis of Gonatopus flavifemur special genes. (padj < 0.05). Supplementary Table 12. Genes under positive selection in Gonatopus flavifemur detected by both aBSREL model in HyPhy and branch-site model in PAML. Supplementary Table 13. GO enrichment analysis of Gonatopus flavifemur expanded gene families, Biological Process category (padj < 0.05). Supplementary Table 14. Venom gland-associated genes (VGGs) in Gonatopus flavifemur. Supplementary Table 15. GO enrichment analysis of VGGs in Gonatopus flavifemur, Biological Process category (padj < 0.05). Supplementary Table 16. Chitinase-like genes in Gonatopus flavifemur and other species. Supplementary Table 17. Neprilysin-like genes in Gonatopus flavifemur and other Hymenoptera genomes. Supplementary Table 18. Upregulated genes in the prey-feeding female transcriptomes compared to the sucrose-feeding female transcriptomes. Supplementary Table 19.Yellow genes and expression level in Gonatopus flavifemur. Supplementary Table 20. Downregulated genes in the prey-feeding female transcriptomes compared to the sucrose-feeding female transcriptomes. Supplementary Table 21. GO enrichment analysis of extremely female-biased genes in Gonatopus flavifemur. Biological Process category (padj < 0.05). Supplementary Table 22. GO enrichment analysis of extremely male-biased genes in Gonatopus flavifemur. Biological Process category (padj < 0.05). Supplementary Table 23. Extremely male-biased genes in Gonatopus flavifemur. Supplementary Table 24. Sample information for sequencing used in this study.Additional file 2: Supplementary Figure 1. Genome size of G. flavifemur estimated by k-mer analysis. Supplementary Figure 2. Flow cytometry histograms of Drosophila melanogaster (A) and G. flavifemur (B). Supplementary Figure 3. Concatenated- and ASTRAL-based phylogenetic trees. Supplementary Figure 4. Rank-based branch length comparison of protein evolution rates in the G. flavifemur branch. Supplementary Figure 5. Venom gland-associated genes (VGGs) in G. flavifemur.Supplementary Figure 6. Multiple sequence alignment of the active site motif (DxxDxDxE) of IDGFs in G. flavifemur and other 12 hymenopterans.Additional file 3. Bacterial contaminating contigs in the G. flavifemur genome assembly.