dataesr/scientific-paragraphs-categorization huggingface dataset:
Gene expressions of the HPI axis were measured only before the salinity challenge and were therefore analysed using model 2. The lmer function from the lme4 package was used for all these mixed models  (Bates et al. , 2014). A PCA was performed on the expression of all genes studied in the brain, pituitary and head kidney using the FactomineR package  (Lê et al. , 2008). The coordinates for each individual on the first 3 dimensions were extracted and evaluated using model 1. The same procedure was performed for the expression of the genes evaluated in the gills using model 2. From the novel environment and hypoxia test, the behaviour data (inter-individual distances, velocity and time spent in the center) were analysed before (<60 min) and after (≥60 min) the hypoxia challenge using a linear mixed model using chronic stress, time (categorical) and their interaction as fixed effects and the day of experiment as random effect.
J o u r n a l P r e -p r o o f Journal Pre-proof required to obtain significances from linear mixed models, might provide small differences between variables measured on the same individuals. When one fixed effect (or the interaction) was found significant, a Tukey post hoc test was completed with the glht function from the multcomp package to test significant differences between the levels of the fixed effect. Finally, correlations between all variables of interest across all individuals were calculated and illustrated using the rcorr and the corrplot functions from the Hmisc and corrplot packages respectively.
Consequences on growth In the present study, the most apparent effects of the chronic stress protocol were detected on biometric results, with a clear reduction in growth of body mass and length. It is well known that stress is energy consuming, decreases appetite through well-described molecular mechanisms and thus reduces growth performances in fish  (Sadoul and Vijayan, 2016). These growth data are therefore not surprising and in agreement with previous studies  (Samaras et al. , 2018; Santos et al. , 2010). Samaras et al. (  2018 ) also observed growth reduction for European sea bass exposed to a "high stress regime" over three weeks, and this reduction led to a 6. 5% difference in final body mass. Based on body mass results (9% difference), the stress protocol (also over three weeks) could be considered to lead to a more severe stress load.
Acknowledgments We are grateful to  Marie-Odile Vidal ,  François Ruelle , and  Alain Vergnet  for hatching and rearing the fish. The authors would also like to thank  Dr. D. Dufour-Rainfray ,  Dr. P. Emond  and  Mr. A. Lefevre  for LC-MS-MS analysis of seabass plasma samples. The authors are also gratful to  Emilie Levavasseur  for the European sea bass drawing in Fig. 1.
Gene expression analysis was performed by quantitative polymerase chain reaction (q-PCR) with the cDNA samples according to the requirements of the MIQE guidelines  (Bustin et al. , 2009) , using the iTAQ Universal SYBR R Green Supermix (Bio-Rad, El Prat de Llobregat, Spain) in Hard-Shell R 384well PCR plates (Bio-Rad, El Prat de Llobregat, Spain) and the CFX384TM Real-Time System (Bio-Rad, El Prat de Llobregat, Spain). The q-PCR program was: 3 min at 95 • C, 39 × (30 s at 95 • C, 30 s at primer melting temperature and fluorescence detection), 5 s at 55 • C followed by a melting curve ranging from 55 to 95 • C with an increase of 0. 5 • C every 30 s. The primer sequences and GenBank accession numbers are shown in the Table  2. The reference genes elongation factor 1 alpha (ef1α), ribosomal protein S18 (rps18), ribosomal protein L27 (rpl27), and mitochondrial import receptor subunit TOM20 (tom20) were analyzed and the combination of the most stable ones was used to calculate the relative expression of the genes of interest following the Pfaffl method  (Pfaffl, 2001).
Acknowledgments DM was funded by the  European Union  and  Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE)  in the framework of the  Marie-Curie   FP7 COFUND People Program , through the award of an  AgreenSkills/AgreenSkills+ fellowship , and by and the  FRAGCLIM Consolidator Grant , funded by the  European Research Council  under the European Union's  Horizon 2020 research and innovation programme  (grant agreement number  726176 ). This work was supported by the  TULIP Laboratory of Excellence  ( ANR-10-LABX-41 ) and by the  BIOSTASES Advanced Grant  funded by the  European Research Council  under the European Union's  Horizon 2020 research and innovation program  (grant agreement no. 666971). We also thank three anonymous referees for their insightful comments.
EMF mycorrhization rates of P. taeda and P. trichocarpa roots were examined under a dissecting microscope. EMF mycorrhization rate was quantified by counting the% of mycorrhizal root tips for a total of 200 root tips for an individual sample. For plant dry weight measurement, the whole plants were washed with sterile DDI water, wiped with the sterile towel and then oven-dried for 72 h at 60 • C, cooled to room temperature, separated the aboveground and belowground tissues, and then weighed. Metatranscriptomic data was generated for samples taken at the 4-month time period (n ≥ 3 for biological replicates of each plant combinations-details below). To sample roots, tertiary fine roots and root tips of P. trichocarpa and P. taeda were removed with forceps. Adhered soil was removed by shaking and forceps.
Raw reads were deposited in the NCBI Short Read Archive (accession SRP156890). Bioinformatic pipelines FIGURE 2 | Plant biomass (dry weight and standard deviation) of aboveground plant (above) and root tissue biomass (below) for seedling grown under different plant combinations: Populus trichocarpa grown with P. trichocarpa (P. trichocarpa × P. trichocarpa), P. trichocarpa with Pinus taeda (P. trichocarpa × P. taeda), P. taeda with P. trichocarpa (P. taeda × P. trichocarpa) and P. taeda with P. taeda. Significant groupings determined by Tukey test for whole plant biomass for the individual plant species (P < 0. 05, n ≥ 4). Means are marked by the same letter were not significantly different (Uppercase for P. trichocarpa; Lowercase for P. taeda). P. tri = P. trichocarpa. for data assembly, quality filtering, annotation and comparison are described in Supporting Information Text A2.
P. trichocarpa and P. taeda databases were performed to filter out D1/D2 reads belonging to plastid and mitochondrial rRNA genes. To generate Figures  3A, B , the percentage of reads was calculated according to the ratio of reads from individual taxa vs. total rRNA reads obtained from the individual samples (Supplementary Figure  2,  Step 4). The ecological function of each fungal group was assigned according to FUNGuild  (Nguyen et al. , 2016). Differences in community composition among the comparisons were tested using permutational multivariate analysis of variance (PERMANOVA). Results for PERMANOVA were corrected for multiple comparison using false discovery rate (FDR). P-value were calculated based on pseudo-F statistics, and results with P ≤ 0. 05 were considered as statistically significant (Results shown in Figures  4A-D ; Supplementary  Table 3;  and Supplementary Datasets 2B,D ).
Both non-metric multidimensional scaling (NMDS) and PERMANOVA were performed using vegan package version 2. 5. 3 in R (3. 5. 1). The same RNA was used for amplicon sequence analysis targeting fungal LSU rDNA with the primers LROR and LR3  (Vilgalys and Hester, 1990). Details for amplicon sequencing are described in Supporting Information Text A3. To filter out plant reads, remaining unmapped reads (approximately 30-million; Supplementary Figure  2,  Step S6) were mapped onto reference sequences of P. trichocarpa and P. taeda using TopHat and Bowtie2 packages (detailed see Supporting Information Text A2). To determine the genes belonging to root fungi that were differentially expressed across the three combinations of plant partners, we applied a combination of genome-based mapping and de novo approaches to sort out RNA-seq reads belonging to key functional genes of fungi (Supplementary Figure  2 ).
taeda" belong to EMF genes. Compared to "FunGene_P. taeda, " the contigs of "FunGene_P. tri" reference may contain fungal genes with higher diverse ecological function (e. g. , including AMF, EMF, and endophytes). Comparative transcriptomics were furthered applied for the normalized mapped-reads with the DESeq package (Figures  5, 6 ;  Anders and Huber, 2010). Here, the genes of FunGene_P. tri and FunGene_P. taeda were assigned to the function (KOG) of closely relative fungal taxa using BlastX. The annotated protein sequences from 24 fungal genomes (Supporting Information Text A2_4) were used as references for mapping transcripts. These references were selected based upon their phylogenetic association with dominant fungal taxa detected in our dataset (see Figure  3A ) [e. g. , Rhizophagus, Suillus, Rhizopogon, Tuber, Hebeloma, Pezizaceae (Terfezia), Leptosphaeria, Fusarium, Pleurotus, and Glonium], the dominant soil/root microbiomes that were presented in the Populus sites (e.
g. , Cenococcum, Laccaria, Ilyonectria, Mortierella, Atractiella, Umbelopsis, and a dark septate endophyte, Cadophora)  (Bonito et al. , 2014). The sequences of the functional genes were assigned to the predicted trophic designations (e. g. , genes belong to AMF, EMF) (Supplementary Dataset 4). Here, the classification of a gene to the taxa relied on the sequence that was assigned to a protein reference taxonomy via BlastX (Threshold:% ID > 50, e ≤ 10 -8 , alignment length > 50). The representative ecological group of each assigned gene was further designated based on the predicted ecological function of the assigned reference. The R-scripts used for this study can be accessed through: https://github. com/NFREC-Liao-Lab/Neighbor_plant_ microbiome_RNA.
trichocarpa root tips colonized by EMF  (Baum et al. , 2002; Danielsen et al. , 2013). Over 80% root tips of the Pinus seedlings were colonized with well-formed ectomycorrhizae (Figure  1d  and Supplementary Table  1 ). In contrast, less than 2% of P. trichocarpa root tips showed visible mycorrhizas. Of interest, growing P. taeda near P. trichocarpa did not induce ectomycorrhizas on Populus or change EMF mycorrhization rates of either plant species. The result implies that many   3  and Supplementary Datasets 2B,D). AMF, arbuscular mycorrhizal fungi; EMF, ectomycorrhizal fungi; Sap, Saprotrophic fungi. The pairwise comparisons were applied to each microbial taxon (Supplementary Datasets 2B,D). P. tri = P. trichocarpa. Pinus-associated EMF may not be able to form mycorrhizae on Populus roots.
Identification of Molecular Activities of Plant Root Microbiomes Through Metatranscriptomics We employed metatranscriptomics to examine root tips of P. taeda and P. trichocarpa grown in combinations of heterospecific and conspecific plant bioassay treatments (Figure  1a ). For P. taeda, the root metatranscriptome of Pinus grown in Pinus forest soil exhibited a fairly constant proportionality of reads belonging to fungi and P. taeda. The% reads mapped to fungi, P. taeda, and others (reads not mapped to genome references) were 24% (stdev. 3. 2%), 27% (stdev. 1. 9%), and 47%, respectively (Supplementary Dataset 1). In contrast, fewer fungal reads were recovered from Populus roots compared to Pinus root systems. On average, the% reads mapped to fungi, Populus, and others (reads not mapped to genome references) were 5% (stdev 0.
4%), 59% (stdev 1. 6%), and 37%, respectively. Excluding ribosomal RNA reads (see next section), 37% (from Pinus root samples) and 47% of the reads (from Populus root samples) did not map to fungal genomes or plant transcriptomic database. Some of these reads may belong to plant and fungi, however, were not mapped due to lower abundance of these genes or genome variation within a species. Some of the unmapped reads may belong to the fungi which have not been molecular characterized. These sequences were excluded from when grown with P. taeda compared to with P. trichocarpa; (B) FunGene_P. tri on P. taeda roots that were up-regulated (in green) or down-regulated (in purple) when P. taeda was grown with P. trichocarpa compared to with P. taeda. (C) FunGene_P. taeda on P. trichocarpa roots that were up-regulated (in blue) or down-regulated (in red) when grown with P.
taeda compared to when grown with P. trichocarpa; (D) FunGene_P. taeda on P. taeda roots that were up-regulated (in green) or down-regulated (in purple) when P. taeda was grown with P. trichocarpa compared to when grown with P. taeda. Data of loading gene factors were generated using coordinate scales on the left (log10 of expression rate) and the bottom (mean of log2-fold changes). Cross-comparative expression of the genes was analyzed using t-test to compare the fungal genes of the roots from a plant species grown with conspecific vs. heterospecific neighbors (n ≥ 3; Wilcox. Test P < 0. 01; FDR < 0. 05; fold changes ≥ 2). The total counts of genes detected and significant changes in gene counts in response to neighbor effects are listed in Supplementary Dataset 3. the analysis. For example, relatively few full genome sequences are publicly available to serve as the databases for AMF and EMF species, indicating that some of the excluded reads might belong to the unknown taxa of mycorrhizal fungi.
The attempts to identify the expression pattern of mycorrhizal fungi and other root microbes can be limited by the limitation of current available genome databases. A portion of reads belonging to rRNA were recovered from RNA-seq data using poly-A enrichment strategy for cDNA library construction. In average, 53,943 fungal LSU D1/D2 reads and 144,013 bacterial 16S rRNA reads were recovered from each sample (Supplementary Dataset 1). These recovered reads allowed us to estimate fungal community structure from each sample based the transcriptomic abundance of prokaryotic and eukaryotic rRNA  (Liao et al. , 2014). Since rRNA is one of the basic components of microbial cells, it is likely to be an ideal indicator for principal activity (e. g. , protein synthesis) of fungi and bacteria. The average ratio of fungal: bacterial rRNA in root tissues was around 1.
5:1 (Supplementary Table  2 ). Growing Pinus and Populus together resulted in an increase of fungal-tobacterial rRNA ratio to 2:1 in both plant species (Supplementary Dataset 1). The ratio of fungi:bacteria rRNA reads and the number of fungal species per root sample increased in heterospecific treatments (Supplementary Table  2  and Figure 3 ). Previous study showed that higher fungal:bacterial ratio may lead to higher soil carbon storage and nitrogen allocation to plant species  (Malik et al. , 2016)  and result in facilitating plant growth promotion. It is also possible that heterospecific plant species may enhance the complexity of root exudates or elicitors released in the shared soil ecosystem  (Grayston et al. , 1997). Color-filled circles show loadings of whole gene groups for root samples across different host combinations [P.
trichocarpa (P. trichocarpa × P. trichocarpa) in red; P. trichocarpa (P. trichocarpa × P. taeda) in Blue; P. taeda (P. trichocarpa × P. taeda) in green; P. taeda (P. taeda × P. taeda) in purple; n ≥ 3). The distance between points approximates gene expression pattern differences among the samples. The percentage of variances were indicated in the parentheses. Root-microbiome genes were sorted using de novo assembly. Computational workflows for data analyses are shown in Supporting Information Text A2, Supplementary Figures  2, 3 , and Supplementary Dataset 3. The greater chemical diversity of root exudates may positively correlate to the diversity of microbes recruited by the roots  (Steinauer et al. , 2016). Our data showed that the fungal/bacterial rRNA ratio quickly responded to the plant treatments (4-month after bioassay).
, 2019). A small portion (<5% of total fungal rRNA D1/D2 reads) of transcriptomically active pathogenic (Sporisorium) and saprotrophic (Cladosporium) fungi were also detected in ectomycorrhizal roots of Pinus. Pairwise comparisons between P. trichocarpa (P. trichocarpa × P. trichocarpa) vs. P. taeda (P. taeda × P. taeda) suggested that Populus harbored a higher relative abundance of six AMF taxa (Claroideoglomus, Diversispora, Glomus, Otospora, Septoglomus, and Scutellospora), four EMF taxa (Hyaloscypha, Hymenogaster, Pezizaceae, and Sarcosphaera), and 12 other fungal taxa with endophytic, pathogenic or/and saprotrophic activities (P < 0. 05, Supplementary Dataset 2B); Pinus were dominated by three fungal taxa: Rhizopogon, Marcellenina, and Cladosporium. Overall, the diversity of microbial taxa was distinct in the roots of different plant species, even when the plants were grown in the same soil type.
Plant host species was shown to influence the colonization ability of specific EMF species. These results indicate that host preference plays a substantial role in the composition and rRNA-associated metabolic activities of EMF and some other specific taxa on Populus and Pinus. On average, 0. 5% of the total RNA-seq reads that belong to 16S rRNA from the individual root samples were recovered, accounting for 144K reads in average (Supplementary Dataset 1). A total of 196 bacterial families were detected in roots of P. trichocarpa and P. taeda (Figure  3B  and Supplementary Dataset 2C). When grown with a conspecific neighbor, P. trichocarpa interacted with broad diversity of bacteria and were composed of 5 major orders and 25 major families. These included members of Actinomycetales (30% of total 16S reads), Pseudomonadales (14%), Burkholderiales (9%), Rhizobiales (8%), and Sphingomonadales (5%).
However, the community structures of certain fungal and bacterial taxa can be significantly affected by the species of neighboring plants (P < 0. 05, Supplementary Tables 3B,D). For example, the relative abundance of some fungal taxa increased (Suillus, Rhizopogon) or decreased (15 taxa) when P. trichocarpa was grown with P. taeda compared to when P. trichocarpa was grown with a conspecific neighbor (P < 0. 05; Supplementary Dataset 2B). Although Pinus roots were still dominated by their own associated EMF (e. g. , Suillus, Rhizopogon) while growing with P. trichocarpa, a few P. trichocarpa-associated EMF (Hebeloma, Hyaloscypha, Hymenogaster, and Sarcosphaera), AMF (Claroideoglomus, Diversispora, and Rhizophagus) and endophytes (Tetracladium and Chalara) and other five taxa increased their relative abundance of rRNA on these Pinus roots (Figure  3A ; P < 0.
05, Supplementary Dataset 2B). These results imply that EMF hypha extending from a compatible host root (the mycorrhizal root) are able to reach and interact with heterospecific plant roots even when the second host appears to be incompatible based on morphological development  (Liao et al. , 2016). Also connected by CMNs, in this instance, there was no evidence of mycorrhizal development on roots of the incompatible Populus host (Supplementary Table  1 ). To verify the consistency between key dominant taxa detected in RNA-seq data compared to next generation amplicon sequencing data, cDNA synthetized from rRNA from the same RNA extraction were generated from a subset of samples to obtain full sequences of the LSU D1/D2 region (Supplementary Figure  4 ). Fungal composition detected by cDNA amplicon sequencing targeting rRNA revealed similar patterns as RNA-seq data.
When growing near Pinus, the Populus samples harbored a greater amount of AMF and endophytes (Leotiomycetes) compared to Pinus roots. Boletales (particularly Suillus and Rhizopogon) were consistently abundant in Pinus, which dominate the Pinus rhizosphere as uncovered by RNA-seq data (Figure  3  and Supplementary Figure  4 ). Fungal reads of Tuber spp. (Pezizales) were abundant in RNA-seq data of Pinus but not in cDNA amplicon sequencing data. RNA-seq reads assigned to Suillus were examined by aligning to Suillus reference sequences (D1/D2 regions obtained from fruiting bodies). Results confirm that merged short reads could be aligned to Suillus reference sequences, mostly along the conserved area between D1 and D2 regions (Supplementary Figure  5 ). Bacterial rRNA diversity also varied between plant hosts.
Plant Partners Are Responsible for Shaping Gene Expression of the Root Microbiomes In order to study functional dynamics of microbiomes across multiple plant species, we conducted paired-comparisons for the expression patterns of fungal genes from individual root samples (Figures  5 6 7 ). Specifically, the expression of the gene candidates for "Fungal genes originally expressed in P. trichocarpa roots" (FunGene_P. tri) and "Fungal genes originally expressed in P. taeda root" (FunGene_P. taeda) were compared. Comparative transcriptomics was further analyzed for the normalized mapped-reads with the DESeq package (Figures  5, 6 ;  Anders and Huber, 2010). Many genes from both gene groups (FunGene_P. tri and FunGene_P. taeda) were detected on P. trichocarpa and P. taeda roots (Figure  5 ). Expression patterns of fungal functional genes clustered primarily by plant species (Figure  6 ), indicating that the microbial shifts caused by heterospecific plant neighbors (Figure  4 ) could cause changes in microbial gene expression.
trichocarpa. Future studies are needed to understand whether rhizodeposits from P. trichocarpa structure the molecular function of Pinus EMF specialists. In general, similar proportions of genes for biological process (e. g. , Zn-finger proteins, transcription initiation factors, ribosomal proteins) were enriched across different groups of fungi when grown with heterospecific plants compared to conspecifics (Supplementary Dataset 4). However, functional annotation of fungal genes indicates that EMF specialists enriched for specific molecular mechanisms in response to heterospecific plant species, only when these fungi were grown with their own host plants. For example, Suillus and Rhizopogon were both enriched for genes encoding WD-40 repeats (12% of total enriched gene counts) and cytochrome P450 (3% of total enriched gene counts) when interacting with heterospecific hosts compared to conspecifics, but only when these fungal taxa colonized Pinus but not Populus (Supplementary Dataset 4).
Previous studies on Suillus-Pinus ectomycorrhizal inoculation and metatranscriptomics showed that genes for WD-40 repeat and cytochrome P450 are involved in host recognition  (Liao et al. , 2016). Thus, the molecular mechanisms involved in EMF-host recognition/symbiosis may also be altered when a non-host plant is growing nearby. Suillus and Rhizopogon also expressed higher proportions of transporter genes (0. 6 to 0. 9% of significantly expressed genes) during interaction with heterospecific plants compared to conspecifics (Supplementary Datasets 3, 4). For example, the expression of 401 genes of transmembrane proteins assigned to Suillus and Rhizopogon were significantly increased   4 ). The color bar on the right side of each heatmap [as well as in (B)] represents the numbers of genes assigned to predicted fungal taxa through blast (BlastX) to the fungal protein databases (method in Supporting Information Text A2).
Data that were used to generate heatmaps are presented in Supplementary Dataset 3. Significant changes of gene expression were clustered according to ecological functions of fungal taxa, including AMF, EMF, Pathogens, Saprotrophs, and endophytes (>2-fold changes; FDR < 0. 05). The color key represents RPKM normalized log2 transformed counts of the genes. Wilcoxon signed-rank test  (Bauer, 1972)  was applied to filter data. (B) Fisher's test was used to identify enrichment of genes from the individual taxa underlying 2 × 2 matrix. Boxes in blue showed the genes of the individual taxa were enriched in "I" vs. "II. " Boxes in orange showed the genes of the individual taxa were enriched in "II" vs. "I" (P < 0. 05). in P. trichocarpa (P. trichocarpa × P. taeda) compared to P. trichocarpa (P. trichocarpa × P.