dataesr/scientific-paragraphs-categorization huggingface dataset:
g. secateurs, pruning saw, forceps) should be avoided; this is of particular importance when sampling for pathogens. 4. The best period for sampling varies according to the affected tissues and the suspected causal agents. If possible, at least three samplings per year (spring, summer and fall) should be conducted. 5. Samples should also be taken from apparently healthy tissue to know what healthy plant tissue looks like during normal growth, to potentially detect differences in microbial community composition between healthy and symptomatic tissues, and to study latent infection or endophytes. 6. Proper labelling of sampled material is an essential step without which biological specimens lose their scientific value  (Krogmann and Holstein 2010). The minimal data recorded should include locality, GPS coordinates, host plant, date of collection, collector name, and unique identifying number.
2014). Rapidly evolving high-throughput sequencing (HTS) technologies enable simultaneous identification of thousands of organism species from numerous and complex samples, with protocols available for viruses, bacteria, fungi, oomycetes and animal pests  (Abdelfattah et al. 2018; Tedersoo et al. 2018 ). The available HTS platforms and details for analysis steps are outlined in  Tedersoo et al. (2018). Selecting molecular markers of enough resolution, primers of high affinity to templates, negative and positive control samples and reliable reference sequence databases are the most important factors for HTS-based pest and pathogen identification  (Tedersoo et al. 2018). Correct reference data are critical in the precise identification of plant pathogens and, at present, not all publically available databases are sufficiently accurate to enable accurate identification  (Jayasiri et al.
2015). Thus, it is crucially important to improve and correct pest and pathogen sequences in publicly databases  (Nilsson et al. 2014)  Third-generation sequencing technologies such as PacBio (www. pacificbiosciences. com) and Oxford Nanopore (www. nanoporetech. com) present the possibility to sequence long reads. These technologies have not yet been used in sentinel systems. The benefits arising from amplifying other regions (with sequences longer than ITS1 or ITS2), that could give better identification at the species level, are countered by the absence of adequate reference databases to blast the result obtained. Moreover, these sequencing technologies currently have higher error rates compared with Illumina  (Weirather et al. 2017). Despite this problem, it is necessary to emphasize that the new HTS system, such as the MinION device from Oxford Nanopore has great promise as a useful tool in field applications since its portability allows for in situ (on-site) analysis and real-time data generation, thus making the workflow fully versatile.
The use of HTS platforms for biosecurity purposes such as identifying latent or potentially opportunistic pathogens in asymptomatic host tissues requires some consideration of the technological limitations, including the quality of data output (e. g. Illumina MiSeq). While bioinformatics processing can provide useful data output for biodiversity studies (e. g. metacommunity analysis), blast searching of filtered sequence data against custom or public databases generally results in a limited number of identified species, but with many OTUs assigned to higher taxonomic levels. This problem arises due to following reasons: 1) the low power of single-marker short sequences in differentiating taxa, 2) the low taxonomic coverage of databases, and 3) sequencing errors accumulated in the output reads (the sum of amplification and HTS errors).
2010; Kirichenko et al. 2015). As for pathogens, one of the limitations of DNA barcoding is the lack of appropriate reference databases, which would cover all formally described arthropods. To date, comprehensive databases have been accumulated mainly for certain insect taxa (e. g. Lepidoptera and Coleoptera on http://www. boldsystems. org/; Ratmasingham and Hebert 2007), whereas other groups of arthropods remain underrepresented. In the existing databases, inaccuracies may also appear which can lead to misidentification. The quality and accuracy of the sequences stored in the genetic databases might not always be satisfactory, especially considering that any user can access and add sequences  (Hebert and Gregory 2005). In a recent survey of insects that colonized a sentinel plantation in China, DNA barcoding enabled to reliably identify only one quarter of sample insect species  (Roques et al.
2015)  For nematodes, several genes are targeted for identification such as the mitochondrial cytochrome b locus (mtDNAcytb)  (Mattiucci et al. 2003) , the gene encoding the mitochondrial cytochrome oxidase 2 (COX2)  (Valentini et al. 2006 ) and the mitochondrial cytochrome oxidase 1 (COXI)  (Blouin, 2002) , the ribosomal RNA of the small (ssrRNA) and large subunit (lsrRNA)  (Hu et al. 2001). Other nuclear genes were also selected such as the internal transcribed spacer 1 (ITS1) of rDNA to identify Strongylidae and Anisakidae  (Roeber et al. 2013). NEMBASE (http://www. nematodes. org/nembase4), a publicly available database, provides access to sequences and associated meta-data on parasitic nematode expressed sequence tags  (Elsworth et al. 2011). WormBase is an international consortium of biologists and computer scientists dedicated to the research community and providing accurate, current, accessible information concerning the genetics, genomics, and biology of Caenorhabditis elegans Maupas and related nematodes (http://www.
In contrast, the focus for sentinel nurseries and sentinel arboreta should be on identifying a large number of taxa associated with the host irrespective of whether they are causing damage. HTS technologies are and will continue to play a pivotal role in the study of biological invasions. In sentinel systems, HTS can help filter information on pest or pathogen taxa so as to focus the sampling efforts and identification only on target species. DNA barcoding and metabarcoding are powerful tools that can give an early warning and confirmation of potential causal agents of damage and can permit the study of the microbial community associated with woody hosts to ascertain the origin and functional role of individuals in different environments. However, reliance on HTS data must be weighed against the accuracy of bioinformatics analysis and depth of the sequence database; and be cognizant on what constitutes a positive or negative result  (Martin et al.
Acknowledgements This work was supported by  COST Action Global Warning  ( FP1401 ). DLM and YB contribution was also supported by the  Russian Foundation for Basic Research  (Grant No. 17-04-01486 ). MG was supported by  Ministry of Education, Science and Technological Development of the Republic of Serbia , Grant  III43002. MKA was supported by the  Ministry of Science and Higher Education of the Republic of Poland. NK was supported by  Le Studium foundation (France)  and  RFBR  (Grant No. 19-04-01029 ). RE, IF and MK contribution was also supported by  CABI  with core financial support from its member countries (see http://www. cabi. org/about-cabi/who-we-work-with/key-donors/for details). IF contribution was further supported through a grant from the  Swiss State Secretariat for Science, Education and Research  (Grant  C15.
Preparation and study of the fossil The amber piece has been polished, then encased in a block of polyester resin (see detailed method in  Hoffeins, 2001). This block has been further polished to facilitate the dorsal view of the ant, and is now measuring 18 × 12 × 7 mm. The ant is exquisitely preserved, although missing the right antenna, right foreleg, left mid and hind legs as well as the apical tarsomeres of all legs. It is preserved with a mite, two ant eggs, and several plant debris including stellate hairs as syninclusions. Observations, measurements, and photographs were made using a Leica MZ APO stereomicroscope equipped with an ocular micrometer and a Canon 5D Mark II camera. Helicon Focus 6. 7 software was used to build composite images from a series of photographs taken at different focal planes.
4. 3 (R Development Core Team, 2017), we ran generalized linear mixed models (GLMMs;  [46] ) with binomial error structure and logit link function  [47]  as the output considered was whether the behaviour of interest was observed or not (e. g. box visited from the clumped distribution or not; change of the distribution or not; box baited or not; for the model formulae see table  1 ). Specifically, the linear models aimed to complement the analysis of the Index of Lateralization by better qualifying and quantifying the strategy that was used. Namely, we constructed four types of models that we ran on subsets of our data considering specific tasks/seasons at a time. With a first model ('First visited box') we assessed whether the decision to forage was made initially by investigating whether the first visited box belonged to the clumped distribution or not.
For instance, individuals could target the closest neighbouring box, or rather prefer to leave for the opposite distribution after having fed on a given box. To depict this conditionality, we included in the model only data from the second visited box to the sixth one, but included the 'location of the previous box' (i. e. from the clumped or scattered distribution) as a tested variable (i. e. first order conditionality, see models' description and formula in table  1 ). We considered only till the sixth visited box since each circular distribution contained only six boxes and we assumed that an omniscient individual would forage consecutively on the six boxes from the clumped distribution. Once again, this model was conducted on the two CvS tasks/seasons together (CvS1 and CvS2; N visited boxes ¼ 2466), and separately on the dataset from CvS1 and CvQ pooled together (N visited boxes ¼ 2841).
Models implementation GLMMs were fitted using the glmer function from the lme4 package  [53]  while variance inflation factor (VIF;  [54] ) assessments were conducted using the function vif from the car package  [55]. Correlation, assessed on equivalent models not including random effects (i. e. GLM), was not an issue (VIF max , 2 among all models, that maximum being reached for 'Goal-directed strategy' model, see electronic supplementary material for investigations on this specific case; for the other models it ranges below 2). To ease the interpretability of the results by allowing to have comparable estimates  [56]  we scaled all the continuous predictors. Only the number of competitors was left skewed due to zero inflation, but no transformation allowed reaching a better symmetrical distribution, except doing only a binomial variable, being null if no competitor, or one if at least one competitor was present.
Acknowledgements. We are sincerely grateful to  R. Cozzolino  ( Ethoikos Foundation ), to the  UMR 7206 Eco-anthropologie (Museum national d'Histoire naturelle-CNRS-Univ. Paris 7), to the  SocieteFrancophone De Primatologie (SFDP)  and to the  Primate Center of Strasbourg University  for financial support. Thank you to  Y. Larmet  and his team for allowing us to conduct the study at the  Primate Center of Strasbourg University. We also thank  J. Wittemer  for logistical aid, all caretakers and veterinarians for their helpful assistance throughout the study, the numerous trainees that helped with the data collection (A. Sotto-Mayor, P. Iban ˜ez de Aldecoa ,  L. Berrod ,  L. Marmol-Gomez ,  P. Meunier ,  M. L. Poiret ,  Y. Chantrel ,  C. Lepas ,  M. Bey ,  A. Navarra ,  M. Hirel ,  T. Leclere  and  I.
In the genome of icheumonid wasps producing virus-derived particles, the genes putatively derived from the viral ancestor are located in genomic regions named IVSPERs (for Ichno-Virus Structural Protein Encoding Regions;  [18] ). All IVSPER genes are expressed specifically in the calyx of female wasps, and a number encode proteins that were detected by mass spectrometry analysis of purified IV particles  [18, 20]. However, because they have no similarities in databases, the functions of these genes have remained unknown. Given that particles are produced exclusively in wasp calyx cells and cannot replicate, their morphogenesis cannot be studied in the usual manner, i. e. by injecting virions into a permissive host or infecting appropriate cell lines. Accordingly, viral particle production can only be examined in situ, by studying what occurs in the parasitoid ovary.
dsIVSP4-1 or dsU23 injection impairs nucleocapsid assembly IVSP4-1 and U23 code for unrelated proteins of 446 and 415 amino acids, respectively. In wasps injected with dsIVSP4-1 or dsU23, calyx cells nuclei contained no distinguishable subvirions but abundant membranes, either positioned adjacent to the virogenic stroma, or vesiculated and free in the nucleoplasm (Fig 4A  , 4B  and 4C ). The membranes formed "defective particles" since, although lacking nucleocapsids, they exit the nucleus (Fig 4A ), traffic across the cytoplasm and bud into the oviduct lumen (Fig 4D ), as would normal subvirions. The defective particles appeared to contain small amounts of material situated on the inner side of their inner envelope. To check for the presence or absence of DNA, particles were collected from wasps injected with dsGFP, dsIVSP4-1 or dsU23 respectively and used in PCR reactions with primers specific to GlyPro2 (GenBank: AIK25735), a gene encoded by a packaged H.
Discussion Polydnaviruses are unique virus-derived particles produced by parasitic wasps that rely on them to successfully develop in their insect hosts; however, a viral origin for these particles has only recently become clear. The two recognized PDV taxa, the BVs and IVs, are today known to have originated following the acquisition of two different viral genomes by ancestral parasitic Hymenoptera  [24]. The BVs, so named because of their association with braconid microgastrine parasitoids, derive from a captured nudivirus genome  [17] , and are now carried by thousands of wasp species. The IVs, carried by large numbers of ichneumonid parasitoids, originated following the integration of an extinct or currently unknown viral genome  [18]. While the function of BV genes descending from the nudiviral ancestor can be inferred by virtue of their homology to core nudivirus genes, the function of IVSPER-located IV genes has been unclear, due to the absence of homologous sequences in current DNA databases.
In the present study, we have used RNA interference combined with TEM in an attempt to identify putative IVSPER functions. We initially searched for genes that were upregulated in stage 3 pupae, when the first particles are observed in calyx cell nuclei. In several viruses, including baculoviruses  [25]  and nudivirus-derived BVs  [17, 26] , the temporal expression pattern of viral genes matches well with their function in the viral cycle: "early" transcribed genes mainly encode proteins involved in the replication of the viral genome and/or in the regulation of viral gene expression, whereas "late" genes mostly encode structural proteins (capsids and/or envelope proteins). We thus selected six IVSPER genes meeting the criterion of late expression and which in addition were predicted to encode virion structural proteins  [18].
Complete nucleocapsid assembly is only observed when the expression of IVSPER genes U23 and IVSP4-1 is not impaired. When their expression was inhibited, only empty envelopes were produced. Our work established that following knockdown of either of the two genes, circular DNA species were available within calyx cell nuclei, but were not encapsidated. Many viruses assemble their capsids around their genomes while others, such as baculoviruses and herpesviruses, form first a capsid shell and then package their genome thanks to viral packaging motor proteins  [36, 37]. In H. didymator, empty capsids have never been observed in association with virion morphogenesis, suggesting that DNA packaging probably occurs very rapidly or concomitant with capsid assembly. Whether these two proteins, and particularly U23 which co-localizes with nucleocapsids, are truly structural nucleocapsid proteins, or are necessary to initiate DNA packaging and nucleocapsid assembly, remains to be determined.
Reverse-transcriptase quantitative real-time PCR (RT-qPCR) For RT-qPCRs, 400 ng of purified RNA were reverse-transcripted with the SuperScript III Reverse Transcriptase kit (Life Technologies) and oligo(dT)15 primer (Promega). The mRNA transcripts level of selected IVSPER genes was measured by qRT-PCR using a LightCycler 480 System (Roche) and SYBR Green I Master Mix (Roche) and was normalized relative to a housekeeping wasp gene (elongation factor 1 (ELF-1)). Each sample was evaluated in triplicate. The total reaction volume per well was 3μl including 0. 5 μM of each primer and cDNA corresponding to 0. 88 ng of total RNA and the program for amplification was 95˚C for 10 min, followed by 45 cycles of 95˚C for 10 s, 58˚C for 10 s, and 72˚C for 10 s. Primers used are listed in S5 Table.
RNA-seq analysis Total RNAs were extracted from pools of 40 to 50 ovaries dissected from females at pupal stages 1, 2 and 3. Three replicates were collected for each pupal stage. DNase-treated RNAs were then sent to the Genewiz company (https://www. genewiz. com) for sequencing (1x50bp SR, HiSeq2500). A total of ~2. E+07 reads (~1000 Mbase) were obtained per sample. Reads were mapped on the wasp genome using STAR aligner version 2. 5. 2a [44]  with the default parameters except the following parameters: outFilterMultimapNmax = 5, outFilterMis-matchNmax = 3, alignIntronMin = 10, alignIntronMax = 50000 and alignMatesGapMax = 50000. Read counting was performed with the tool FeatureCounts version 1. 5. 0-p3 [45]  with the default parameters except the following parameters: -g Parent, -M-fraction, and analyzed with package EdgeR  [46].
Transmission electron microscopy Ovaries were dissected from adult wasps between 2 and 3 days after emergence, and treated as described in  [47]. At least three females (taken at different microinjection dates) were observed for each tested dsRNA to ensure consistency of the observed phenotype. For TEM observations, calyces were fixed 2 hours in a solution of 2% glutaraldehyde in PBS and post fixed in 2% tetroxide osmium in the same buffer for 1 h. Tissues were then bulk-stained for 2 hours in 5% aqueous uranyl acetate, dehydrated in ethanol and embedded in EM812 resin (EMS). Ultrathin sections were double stained in Uranyless (DeltaMicroscopy) and lead citrate and examined under a Jeol 1200 EXII electron microscope at 100 kV (MEA Platform, University of Montpellier). Images were taken with an EMSIS Olympus Quemesa 11 Mpixels camera and analyzed with ImageJ software  [48].
Semi-thin sections Ovaries were dissected out of dsRNA treated female wasps then fixed in 4% PFA, dehydrated in ethanol and then embedded in Unicryl resin (EMS) for semi-thin sections. Sections were stained with 1% toluidine blue in 1% sodium borate, or DAPI (4',6-diamidino-2-phenylindole) at 1μg/ml, for observation by light and fluorescence microscopy, respectively. Five different females, taken at 2 different microinjection dates, were observed for each tested dsRNA to ensure reliability of the observed phenotype. Fluorescence was recorded using a laser scanning confocal microscope (Leica microsystems TCS SPE) on MRI platform (University of Montpellier). Images were taken using the software LAS AF (Leica).
For immunofluorescence, semi-thin sections of calyces were incubated with rat anti-U23, rabbit anti-IVp53-2 or guinea pig anti-IVp12-1 (1:1000) sera in PBS containing 0. 1% bovine serum albumin (BSA) and overlaid with Alexa fluor 594 goat, anti-rat IgH (Invitrogen), Alexa fluor 488 donkey, anti-rabbit (Invitrogen) or Alexa fluor 647 goat, anti-guinea pig (Abcam). After each step, semi-thin sections were washed repeatedly with PBS and finally stored at 4˚C. Fluorescence was recorded using a laser scanning confocal microscope (Leica microsystems TCS SPE) on MRI platform (University of Montpellier). Images were taken using the software LAS AF (Leica).
Immunolabeling For TEM immunogold labeling, calyces were treated as described in  [26]. Reaction of the anti-IVp12-1, anti-U23 and anti-IVp53-2 sera were visualized by binding of anti-rat (10nm diameter gold beads), anti-rabbit (20nm diameter gold beads) and anti-guinea pig (10nm diameter gold beads) antibodies (Aurion EM reagents). Ultrathin sections were examined under a Jeol 1200 EXII electron microscope at 100 kV (MEA Platform, University of Montpellier). Images were taken with an EMSIS Olympus Quemesa 11 Mpixels camera and analyzed with Image J software  [48].
PCR-based detection of packaged DNA HdIV particles were purified from calyces of 2 day-old wasp females as described in  [49]. To avoid contamination with non-packaged DNA, particles were submitted to a DNase treatment using the Turbo DNA-free kit (Life Technology/Invitrogen) according to the manufacturer's protocol. DNA was subsequently extracted from the particles according to standard protocols  [49]  and used as a template for PCR amplification of the HdIV gene GlyPro2 (GenBank: AIK25735. 1). The IVSPER gene U23 and the wasp COI gene were used to control absence of genomic contamination in the samples (primer sequences are available in S5 Table ). The control consisted of DNA extracted from HdIV particles collected from untreated females and not subjected to DNase treatment.
PCR-based detection of circularized HdIV DNA Genomic DNA was extracted from isolated ovaries dissected from 48 h old females, using the QuickExtract T Bacterial DNA extraction Kit (Epicentre/TEBU-BIO) according to the manufacturer's instructions. Genomic DNA extracted from males was used as a negative control for viral DNA circularization. Genomic DNA was used as a template for PCR amplification using primers specific to HdIV segment 29 (Hd29, GenBank: KJ586303. 1) and using primer pairs that were designed for amplification of either the integrated sequence (HdBR-RR/HdBR-RL) or the episomal Hd29 sequence (HdBR-LL/HdBR-RR) (primer sequence in S5 Table ).
qPCR data analysis Data were collected using Light-Cycler 480 software. PCR amplification efficiency (E) for each primer pair was determined by linear regression of a dilution series (5x) of cDNA pool. Relative expression using the housekeeping gene ELF-1 as a reference was calculated using advanced relative quantification (Efficiency method) software provided by Light-Cycler 480 software  [50]. For statistical analyses, Levene's and Shapiro-Wilk tests were used to verify homogeneity of variance and normal distribution of data among groups tested. Differences in gene relative expression between the 4 developmental stages and between dsGFP and dsRNA injected females were tested using two-tailed unpaired t-test for group comparison. When homogeneity of variance was not assumed, Welch-test was used to compare gene relative expression between groups.
Fig). Absence of packaged DNA in purified "defective particles" was also confirmed by the lack of DAPI labeling in the calyx lumen of treated females compared to controls where calyx fluid was intensively stained (S2B Fig). The circular DNA molecules normally packaged into virions result from the amplification and circularization of chromosomal sequences. Absence of DNA within "defective particles" could be caused by the absence of circularized viral molecules. Assessment of the presence or absence of these circular IV DNA molecules in the nuclei of calyx cells of dsU23 or dsIVSP4-1 treated wasps was performed by PCR using ovarian genomic DNA as template and primers amplifying specifically the circular form of the Hd29 molecule (GenBank: KJ586303. 1; see S2C Fig for primers position). Amplification of PCR products in all dsGFP, dsIVSP4-1 or dsU23 wasp samples indicate that circularization of IV chromosomal sequences was not eliminated by knockdown of U23 or IVSP4-1 (S2C Fig).