dataesr/scientific-paragraphs-categorization huggingface dataset:
Aligned sequences were then extracted and realigned on the complete NCBI nr database. The final Blast outputs were filtered to keep only sequences which aligned on sequences of endogenous and exogenous nudiviruses reported in the database. To annotate nudivirus genes on the C. nolae genome, open reading frames were predicted using orfipy (v0. 0. 4)  (Singh & Wurtele, 2021). Nudivirus genes were then annotated by reciprocal blasts using translated sequences from predicted ORFs. Miniprot (v0. 9-0) (H. Li, 2023)  was also used to align C. capitator proteins on C. nolae genome and validate previously annotated nudivirus genes. Miniprot gene models were also used for gene clustering in the dN/dS ratio analysis. Pseudogenized genes were identified by reciprocal blast: tblastn was used first to map nudivirus proteins on the wasp genomes.
Hits were then verified by blastx to map potential nudiviral nucleotide sequences on the NCBI nr database. Proteins corresponding to Nucleotide sequences mapping back on nudivirus were kept and incomplete and non-transcribed sequences were considered as pseudogenes. Ichnovirus sequences were searched using a home-made pipeline where genomic IVSPER sequences of Hyposoter dydimator and Glypta fumiferanae were first aligned on Campoplex genome portions (the genomes were segmented into chunks of 500,000 bases). Aligning chunks were then aligned on the NCBI nr database. These hits were filtered to keep only hits aligning on IVSPER proteins that are absent from parasitoid wasps which do not bear an Ichnovirus. These "IVSPER" hits were then aligned back on the nr database and only the first hit per query was retained.
CO1 and nudivirus gene alignments Because the endogenized nudivirus genomes were highly similar in both Campoplex species, we collected evidences to confirm that the Campoplex wasps sequenced for this study are part of different species. To determine if the genomes of C. capitator and C. nolae come from distinct species, whole genome alignments were performed using D-Genies (v1. 4. 0)  (Cabanettes & Klopp, 2018). A CO1 (cytochrome oxydase 1) sequence alignment was also performed in order to measure divergence between the two genomes. CO1 orthologs were searched for in C. capitator and C. nolae genomes by aligning ORFs on CO1 sequence of Campoplex species reported in BOLD database  (Ratnasingham & Hebert, 2007). Candidates were then aligned together (C. capitator candidates vs C. nolae candidates) before being aligned on the NCBI database.
The tree image was then built using ape (v5. 7-1), treeio (v1. 24. 0) (L. -G. Wang et al. , 2020)  and ggtree (v3. 8. 0)  (Yu et al. , 2017)  packages with R (v4. 2. 1). A whole genome synteny analysis was performed between V. canescens, C. capitator and C. nolae using D-Genies. The cluster synteny figure (Figure  3 ) between C. capitator and V. canescens was built using R and parity plots (Figure  4 ) showing syntenies among nudiviruses were generated with Excel (Microsoft v16). To estimate the evolutionary constraint occurring on groups of genes according to their function, protein sequences of C. capitator, V. canescens and C. nolae were first clustered using OrthoMCL (v2. 0. 9) (L. Li et al. , 2003)  and scripts written by Arun Seetharam (https://github. com/ISUgenomics/common_scripts).
CcapiENV gene expression Paired raw reads from ovaries, head-thorax, testes and venom gland of C. capitator wasps were first trimmed using Trimmomatic (v 0. 38)  (Bolger et al. , 2014)  and TruSeq3-PE adaptors. A quality check was then performed on trimmed reads using fastqc (v 0. 11. 9) before mapping. Reads were mapped on the genome using Hisat-2 (v2. 2. 1-3n)  (Kim et al. , 2019)  after genome indexation. Mapped reads were then sorted using samtools (v1. 16). Sorted reads were converted into counts by FeatureCounts from the Rsubread package (v2. 14. 1)  (Liao et al. , 2019). Using the edgeR package (v 3. 42. 2)  (Robinson et al. , 2010) , genes with a count being equal to less than 15 per sample were filtered from the dataset in order to remove nonexpressed genes. The dataset was then normalised by converting the read counts into TPM (transcripts per million).
5% polyacrylamide gel) under denaturing conditions. Separated bands were incubated for 1h with a Coomassie blue solution (10% acetic acid, 50% ethanol, 0. 1% R250 Coomassie blue, q. s. to 1L of milliQ water) and 9 slices were cut from the gel. Slices were chopped into 1mm 3 cubes before being stored at -20°C and analysed by the platform PIXANIM (France) using a nanoUPLC_LTQ-VElos Pro Orbitrap Mass Spectrometer. For each protein identified by the mass spectrometry, sites and domains were searched for using InterProScan and gene ontology terms were attributed using sequence homology with curated proteins from the Uniprot SWISSprot database (downloaded the 18th November 2022). Proteins with a peptide abundance higher than 50 were then retained as VLP candidates.
Acknowledgements The authors would like to thank  Cosci Francesca ,  Filippo Di Giovanni  and  Augusto Loni  from the  Department of Agriculture, Food and Environment (DAFE) of the University of Pisa (Italy ) for the help provided during field wasp sampling. The authors would like to thank also  Amine Chebbi  who helped the first author on the transposable element annotation pipeline. We would like to thank the  IBiSA Electron Microscopy Facility of the University of Tours  and the  University Hospital of Tours  for their assistance. Research funding was provided by an  ADS Research Grant  from the  Smithsonian Institution  to BFS, who was also supported by a  GGI Peter Buck postdoctoral fellowship  during early stages of this work.
01%. Leydig cells were labelled with a rabbit primary antibody directed against the cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) (1:250, Sigma-Aldrich, Ref HPA016436). Germ cells were stained using LIN28 rabbit primary antibody (1:800, Abcam, Ref Ab462020). Sertoli cells were labelled using AMH goat primary antibody (1:200, Santa-Cruz, Ref sc6886). Apoptotic and proliferating cells were stained with a polyclonal rabbit primary antibody directed against cleaved caspase-3 (1:100; Cell Signaling Technology, Ref 9661), and a monoclonal mouse primary antibody directed against KI-67 (1:100; Dako, Ref M7240), respectively. The appropriate biotinylated goat anti-mouse (Dako, Ref E0433) and anti-rabbit (Vector, Ref BA-1000), and rabbit antigoat (Dako, Ref E0466) antibodies were used at 1:500 as previously described  [29].
Signal corresponding to the secondary antibody was amplified by an incubation with an avidin and biotinylated enzyme complex (ABC Vectastain Elite Kit, Vector Laboratories). Sections were developed using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) and then counterstained with Masson's Hemalun and Lithum Carbonate before dehydration. Between all steps (except between blocking of non-specific sites and the first antibody incubation), sections were washed using phosphate buffer saline (PBS) with 0,1% of Tween 20 detergent (Sigma-Aldrich). The number of LIN28-positive germ cells was determined by counting the stained cells in 8-12 randomly selected histological explant sections of each condition, using a light microscope (Bh2 Olympus Microscope) coupled to Mercator Expert Software (Explora NOVA).
For the 3-day cultures, apoptotic cleaved caspase 3-positive and proliferative KI67-positive cells were counted in the testis cords (including germ and Sertoli cells) and interstitial tissue (including Leydig cells) in 8-12 randomly selected histological explant sections of each condition, giving socalled "intracordonal" and "extracordonal" counting values. The surface area of each section was measured and data presented as cell number per surface area unit. In long-term cultures, relative Leydig and Sertoli cells, and apoptotic cell area were estimated by relating the surface occupied by the CYP11A1-positive Leydig cells, AMH-positive Sertoli cells, and cleaved caspase-3-positive cells respectively onto the total surface of 8-12 randomly selected histological explant sections. For this purpose, we measured the surface occupied by each investigated cell type labelled in dark brown by DAB and related it to the surface of the corresponding explant using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).
Immunofluorescence Sections stained for RNAscope were blocked with PBS-BSA at 10% for 30 min at RT and incubated overnight at 4 °C with primary antibody. Leydig cells were labelled with a rabbit primary antibody directed against CYP11A1 (1:100, Sigma-Aldrich, Ref HPA016436). Germ cells were stained with a LIN28 rabbit primary antibody (1:500, Abcam, Ref Ab46020). Sertoli cells were labelled with an AMH goat primary antibody (1/100, Santa-Cruz, Ref sc6886). Serial frozen sections from MALDI imaging were rehydrated in PBS, submitted to antigen retrieval with citrate buffer as described above, and processed for fluorescent CYP11A1 and AMH double immunostaining. The appropriate AF488 chicken anti-rabbit (Invitrogen, Ref A21441) or AF488 Donkey anti-goat secondary antibody (Invitrogen, Ref A11055) were used at 1:500 for 2 h at RT.
Data preprocessing and normalization A phred quality score higher than 10 was required for the first reads (R1, 16 bases). Among these, the first 6 bases correspond to the unique sample-specific barcode needed to demultiplex the sequencing data, while the following 10 bases correspond to a unique molecular identifier (UMI) used for quantification purposes. The second reads (R2) were aligned to the human reference transcriptome from the UCSC website using BWA version 0. 7. 4. 4 with the parameter "l 24". Reads mapping to several positions in the genome were filtered out from the analysis. After quality control and data preprocessing, a gene count matrix was generated by counting the number of unique UMIs associated with each gene in lines for each sample in columns. The UMI matrix was further normalized with the regularized log (rlog) transformation package implemented in the DeSeq2 package  [34].
Results The endocannabinoid system is dynamically regulated in the fetal testis at 6 to 17 weeks of development The ECS comprises synthesis and degradation enzymes responsible for the regulation of endocannabinoids, AEA and 2-AG (Fig. 1 ) and their receptors CB1 and CB2. The expression of synthetizing/degrading enzymes of AEA (Fig. 1A-G ) and 2-AG (Fig. 1H-N ) was investigated using a previously published RNA-seq dataset  [30]  arising from the analysis of the genome of 6 to 17 DW human testis bulk RNA. During the gestational period spanned, various dynamics of expression were observed for the different enzymes of the ECS. For synthetizing enzymes, the expression of transcripts encoding NAT10 significantly decreased between 6 and 17 DW, whereas the expression of transcripts encoding NAPE-PLD, GDE1 ABHD4, PLGC1, PLCD1 DAGLA, and DAGLB, did not vary significantly (Fig.
2C, D ). As expected, PC (32:0) lipid (PC = phosphatidylcholine; m/z 772. 5254 in green) was observed in testis cords as well as broadly distributed in the interstitial tissue of the hilum, while PC (38:7) lipid (m/z 804. 5531 in dark blue) localized in CYP11A1rich areas of the interstitial tissue and within the testis cords. Consequently, testis cords containing both lipid types appeared in clear blue. In agreement with the above HPLC result showing a low amount of AEA, this endocannabinoid could not be detected by MALDI imaging, as opposed to 2-AG, which was clearly present in the tissue and colocalized mainly with the PC(38:7) lipid, corresponding to Leydig cells area (Fig. 2E, F ). The expression of CB1 (CNR1) and CB2 (CNR2) receptors mRNAs in the fetal testis was investigated using our RNA-seq dataset  [30] , as above (Fig.
The analysis of RNA-seq data additionally highlighted the expression in the human fetal testis of the genes CNR1 and CNR2, which encode the two main cannabinoid receptors, CB1 and CB2. CNR1 expression was variable between 7 and 17 DW and overall higher than CNR2, as confirmed by RT-qPCR. In 10 to 12 DW testis, in situ hybridization using RNAscope, a recognized highly specific and sensitive technique, demonstrated the expression of CNR1 mainly in gonocytes and Leydig cells, and to a lower level in Sertoli cells. Small dots (each dot represent the detection of an RNA copy) corresponding to CNR2 were occasionally observed in these three cell types. Biggest dots were observed in the case of CNR1 meaning the amount of CNR1 RNA is more important. CB1 and CB2 detection in the fetal testis by immunohistochemisty was not successful, which could be due to their relatively low expression levels combined with the high background staining generated by the available antibodies.
Experimental sites, growth and sampling conditions Data were recorded on fruit grown on 18-years-old mandarin trees (Citrus reticulata Blanco cv Willowleaf) of the Inrae-Cirad Citrus Biological Resource Centre of San Giuliano in Corsica (42 • 18 ′ 55"N, 9 • 29 ′ 29"E; 51 m above sea level). Poncirus trifoliata (L) Raf. was used as rootstock for all scions. Trees were grown under the same conditions as previously described by Ref. [14]. Water was supplied every day on the basis 100% replacement of actual evapotranspiration estimated from the Penman-Monteith equation  [25]. Fertilizers were provided to meet standard conditions. Four trees were selected for this experiment and the treatments were randomly distributed in the plot. Only fully expanded flowers from May were selected to obtain fruit of similar age.
Quantitative PCR analysis RNA was extracted from juice sac cells with three biological replicates. First-strand cDNA was synthesized from 500 ng L -1 of total RNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo scientific, Waltman, USA). Primer3 software (ver. 0. 4. 0; http:// frodo. wi. mit. edu/primer3/) was used for primer design. Quantitative PCR was performed on the StepOne-Plus™ (Applied Biosystems, Foster City, CA, USA), using SYBR® Green (Quanta Biosciences, Gaithersburg, USA). A total reaction volume of 15 μL was used. The reaction mix included 2 μL cDNA, 0. 4 μL of reverse primer, 0. 4 μL of forward primer, 10 μL PerfeCTa® SYBR® Green SuperMix ROX™, and 4. 2 μL RNA-free water. A qPCR assay was performed using the following conditions: 95 • C for 5 min followed by 40 cycles of 95 • C for 15s and 60 • C for 40 s.
Statistical analyses All data were analyzed using the R and RStudio statistical software (http://www. R-project. org, accessed on July 20, 2022) and Rstudio (Rstudio Inc. , Boston, MA, USA, version April 1, 1717). All data were expressed as mean values (± standard error) and submitted to two-way ANOVA with an LSD test when a significant difference was detected at p < 0. 05. A two-way hierarchical clustering classification was performed on normalized data (Ward's method) and presented as heat maps, using the gplot package and heatmap. 2 function.
MATERIALS AND METHODS Bacterial strains and growth conditions All tested bacterial strains are listed in Supplementary Dataset S1. The Bradyrhizobium strains were grown in YM or AG medium at 28 °C  [16, 17]. All strains were checked for purity by streaking on agar plate and for identity by ITS sequencing as described  [18]. Escherichia coli strains were grown at 37 °C in modified Lysogeny Broth (LB) medium  [19]. Agrobacterium rhizogenes Arqua1 and Agrobacterium tumefaciens GV3101 were grown at 28 °C on AG medium and Ralstonia pseudosolanacearum was grown in rich BG medium  [20]. When required, the media were supplemented with the appropriate antibiotics at the following concentrations: 50-100 µg/mL kanamycin, 20 µg/mL nalidixic acid, 20 µg/mL cefotaxime and 100-200 µg/ mL spectinomycin.
Genome sequencing and phylogenomic analysis The genomes of strains ORS86, ORS111, STM3557, STM3561, STM3562, STM3566, LMG29515 and LMG09283 were sequenced by combining long read and short read sequencing technologies as previously described  [21] , assembled using the CulebrONT pipeline  [22]  and annotated using the MicroScope platform  [23]. The other genomes downloaded from the NCBI or GenBank databases were annotated de novo by the MicroScope pipeline, and are also publicly available from the MicroScope platform (http://www. genoscope. cns. fr/agc/microscope). From all these genome sequences, a phylogenomic tree was constructed using IQ-tree v2. 2. 0. 3  [24]  based on a multi sequence alignment of 725 genes of the core genome shared by at least 98% of Bradyrhizobium strains, generated by PPanGGolin v1.
Prediction of T3E repertoires, search of SUMO-proteases and phylogenetic analysis The search of T3Es was performed as previously described  [12]. The search for SUMO-Proteases was performed by interrogating the precomputed InterProScan annotations hosted by the MicroScope platform for each genome, using the InterPro identifier (IPR003653) corresponding to the C-terminal catalytic domain of the ULP1 protease family. Protein alignments of ErnA and SUMO-Proteases were performed using ClustalW (http://www. clustal. org/omega/) [29]  and phylogenetic analysis using IQtree as described above.
Plasmid construction, mutagenesis and complementation Standard molecular biology techniques were used for all cloning work. All constructions made in this study are listed in Supplementary Dataset S2, which also includes the primers and the cloning strategies. Briefly, the insertional mutants were obtained by single-crossing over. For this purpose, a 200 to 300-bp internal fragment of the target gene was amplified by polymerase chain reaction (PCR) and cloned into a derivative of the non-replicative plasmid pVO155 carrying an antibiotic resistance marker appropriate to the strain to be mutated (pVO155-npt2-GFP-npt2-Cefo for NAS96. 2 and LMTR13 strains; pVO155-npt2-GFP-npt2-Sp for WSM1744 and ORS86)  [5, 30]. The deletion WSM1744Δsup3 mutant was obtained by double crossing over. For this, 750 to 1000-bp PCR fragments corresponding to the upstream and downstream flanking regions of the sup3 WSM1744 gene (JAAVLW01_10281) were merged by overlap extension PCR and cloned into the pNPTS129 vector, which carries the sacB suicide gene  [31].
For complementation experiments using the ORS3257ΔernA mutant, full-length ernA USDA110 , ernA USDA61 , sup3 WSM1744 , sup3 NAS96-2 and bel2-5 USDA61 were amplified as described above and cloned downstream of the ernA ORS3257 promoter into pVO155-pm-ernA (Supplementary Dataset S2) before re-introduction into ORS3257ΔernA by single-crossing over. For site-directed mutagenesis of ernA, we used the plasmid pVO155-pm-ernA::ernA ORS3257 previously constructed to complement the ORS3257ΔernA mutant  [12] , the Q5 Site-Directed Mutagenesis Kit (NEB) and the primers indicated in Supplementary Dataset S2.
Sup3 secretion analysis Sup3 WSM1744 , GFP and PopP2 coding sequences were introduced by LR recombination in pLAFR6-P2GFH/hrpB vector for expression of the protein of interest fused with a C-terminal 3x-HA epitope tag under the control of the HrpB-activated popP2 promoter sequence (Supplementary Dataset S2). The derived recombinant plasmids were introduced into Ralstonia pseudosolanacearum ΔpopP2 strain (GRS100  [34] ) and ΔpopP2/ Δhrcv  [35]  strains by electroporation and selected on 5 µg/mL tetracycline and 7. 5 µg/mL gentamicin. Production of R. pseudosolanacearum concentrated supernatants (from 10 mL of culture) was performed as previously described  [36]  with minor modifications. Briefly, transformed bacteria were grown in MP secretion medium (MP medium 1×, Congo Red 100 µg/ mL, glutamate 20 mM and tetracycline 5 µg/mL) for 30 h at 28 °C under shaking.
Agrobacterium tumefaciens infiltration assays in Nicotiana benthamiana Binary plasmid pB7FWG2-35S-eGFP-3Flag-sup3 WSM1744 used for constitutive expression of eGFP-3Flag-Sup3 (Supplementary Dataset S2), was introduced into A. tumefaciens strain GV3101 by electroporation  [37]. Agroinfiltration of N. benthamiana leaves and localization of fluorescently labeled Sup3 were performed as previously described  [12].
Hairy root transformation with Agrobacterium rhizogenes Plasmid p35S-sup3 containing sup3 NAS96-2 under the control of the 35S promoter (Supplementary Dataset S2), and the empty vector pJCV51 with the DsRed marker (https://gateway. psb. ugent. be), were introduced by electroporation into the A. rhizogenes Arqua1 strain used for hairy root transformations. A. indica root transformation was performed following previously described procedures  [38].
RESULTS A large diversity of Bradyrhizobium strains nodulate A. indica We inoculated A. indica with 196 Bradyrhizobium strains and found that 69 strains induced nodules or nodule-like structures (Supplementary Dataset S1). Six distinct symbiotic phenotypes (named Type A to F) could be distinguished (Fig. 1 ). The Type A was the largest with 127 strains unable to induce nodules (Nod -). The 30 Type B strains showed a very low nodulation capacity, only a part of the inoculated plants displayed a low number (<5 per plant) of nodule-like structures and infection of these nodules was rare and limited to the intercellular spaces. The 16 Type C strains, including USDA61, induced a large number of nodules in all inoculated plants but the infection remained limited to the intercellular spaces as in Type B.
Type D was observed for only three strains (BR2003, ORS86 and ORS111), and is characterized by a multilobed unorganized nodule development leading to numerous protuberances all along the root, which look more like galls than nodules. The presence of intercellular bacteria could sometimes be detected in these structures. In Type E (11 strains), which includes ORS3257, the elicited nodules were large, generally misshapen but with a clear intracellularly infected central tissue. However, as previously described  [5] , the intracellular bacteria did not differentiate properly into bacteroids. This is correlated with no or very low nitrogenase activity (Supplementary Dataset S1), which in all cases was insufficient to support plant growth (Supplementary Fig. S1 ). Finally, the Type F (9 strains), mainly represented by photosynthetic Bradyrhizobium strains, corresponds to bacteria inducing regular round-shaped nodules, infected intracellularly by bacteria differentiated into spherical bacteroids  [39] , which efficiently fixed nitrogen for the plant benefit.
Colored ranges > Symbiotic phenotypes We found that 45 of the 59 nodulating strains have an ErnA homolog (Supplementary Dataset S3), supporting the idea that ErnA is a widespread T3E in the Bradyrhizobium genus to trigger nodulation. However, correlation between the presence of ErnA and the ability to nodulate is not strict, as 33 strains having a complete ErnA homolog were Nod -and on the contrary, 14 strains lacking an ErnA homolog nodulated A. indica (Fig. 2 ).
Strain NAS96. 2 contains a novel putative ET-Nod As mentioned above, 14 strains lacking an ernA homolog can nodulate A. indica (Fig. 2 ). This raises the question: Do these strains use a T3SS-independent mechanism or an alternative T3E that mimics the action of ErnA? We focused on the NAS96. 2 strain, which is closely related to three strains (SEMIA6148, SEMIA690, BR1035) unable to nodulate A. indica but having a T3SS (Fig. 2 ), suggesting the possibility that NAS96. 2 has a specific ET-Nod that is absent in these three related strains. By comparing their putative effectome (Supplementary Dataset S4), we identified three strain-specific T3Es in NAS96. 2, that we annotated Sup1, Sup2 and Sup3, considering that all of them encode putative SUMO-proteases. Insertional mutants of these three genes were constructed as well as a T3SS mutant (insertion mutant in the rhcN gene).