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This work was undertaken at the University of California Davis Dairy Teaching and Research Facility from June to September 2018. All experimental protocols were approved by and carried out in accordance with the University of California Davis Institutional Animal Care and Use Committee (protocol # 20505).TreatmentsWe enrolled all female calves born between June 19 and September 1 2018, for a total of 28 Holsteins and 8 Jerseys. Our sample size was determined by the availability of calves being born in our herd of approximately 105 lactating cows during this period. Calves were blocked by birth order and randomly allocated to 1 of 3 treatments balanced for breed: disbudded the morning of (Day 0) or 21 days before (Day 21) the startle test, or sham-disbudded (Sham, n = 12/treatment). Among the control calves, half were sham-disbudded the morning of the test, whereas the other half underwent the procedure 21 days earlier. Birth weights were similar across treatments (mean ± SD; Day 0: 35 ± 5 kg; Day 21: 35 ± 6 kg; Sham: 36 ± 9 kg). The startle test occurred between 25 and 32 days of age for all calves. Thus, all Day 0 calves and half of the Sham calves were disbudded between 25 and 32 days of age, and all Day 21 calves and half of the Sham calves were disbudded between 4 and 11 days of age. This design meant all animals were at the same stage of cognitive and motor development during data collection. This was a priority for us because we expected age to strongly influence behavioural responses during the startle test. While it is also possible that disbudding at different ages may affect responses, previous research suggests disbudding has similar outcomes across this range13,15,20.Animal husbandry and housingImmediately after birth, calves were housed individually in outdoor enclosures consisting of a plastic hutch (2.0 m long × 1.5 m wide) and a wire-fenced pen (2.0 m long × 1.5 wide × 0.9 m high). The enclosures were spaced 0.5 m apart and bedded with sand approximately 15 to 20 cm deep.Calves were bottle-fed colostrum twice a day for 5 days. From 5 days of age, calves received milk replacer (26% CP and 16% fat, 15% total solids; Calva Products Inc., Acampo, CA) in bottles at 0645, 1245, and 1845 h. At each meal, Holsteins were fed 1.9 L from 1 to 13 days, 2.4 L from 14 to 23 days, and 2.8 L from 24 days. Jerseys received 1.4 L from 1 to 13 days, 1.9 L from 14 to 23 days, and 2.4 L from 24 days. Water and starter (18.3% CP, 2.8% fat, 4% crude fat; Associated Feed & Supply Co., Turlock, CA) were provided ad libitum in buckets. As part of a separate concurrent study, 11 calves (3 Sham, 3 Day 21, 5 Day 0) received chopped mountain grass hay (34% CP) ad libitum.DisbuddingDisbudding occurred between 730 and 1000 h. For the procedure, the calf was restrained in a head device in her home enclosure21. A 5 × 5 cm patch of hair was clipped with a size 40 electric razor blade on each side of the head to locate the horn bud. We used a 20 gauge × 25 mm needle to administer a cornual nerve block consisting of 5.5 mL buffered lidocaine (2% lidocaine hydrochloride diluted with 8.4% sodium bicarbonate in a 10:1 ratio). If the horn bud was not numb after 10 min, as assessed by pinprick, we gave an additional 2 mL of buffered lidocaine (13% of horn buds). An electric cautery iron (X50, Rhinehart Development Corp., Spencerville, IN) was fitted with a 1.3 cm tip and heated to 439 ± 15 °C (mean ± SD). It was applied to the horn bud for 17 ± 5 s (mean ± SD). Immediately before disbudding, the calf received approximately 1 mg/kg of meloxicam tablets in a gelatin capsule (3.5 g; Torpac Inc., Fairfield, NJ). For Day 0 calves, meloxicam was given after the startle test had occurred later that same day (maximum 12 h later) to ensure the calf was in a drug-free state during the test. Sham-disbudded calves received the same treatment, with the exception that the iron was ambient temperature and the gelatin capsule was empty. Sham calves did not receive meloxicam because the Animal Medicinal Drug Use Clarification Act limits nontherapeutic off-label use of this drug22. SJJA performed all disbudding procedures.ArenaWe tested calves individually in a single 10-min period in a shaded outdoor arena bedded with 10 to 15 cm of sand. The arena was divided into a waiting pen (2.0 × 1.5 m) and a test pen (3.0 × 5.5 m) constructed of 0.9 m high wire panels (MidWest Homes for Pets Foldable Metal Exercise pen, Muncie, IN). A rolling gate provided access between the pens (Fig. 1).Figure 1Aerial view of the arena used for startle tests, including the position of the milk bottle and speaker used to broadcast the startle noise. Figure is drawn to scale.Full size imageA bottle containing 500 mL of the calves’ regular milk replacer was secured to the panel opposite the entrance to the test pen. The bottle was fitted with a rubber teat positioned 80 cm above the ground. Between calves, a fresh bottle was placed in the arena and urine and feces were removed with a shovel.Testing procedureCalves were habituated to the arena for 15 min daily between 700 and 1100 h for 3 consecutive days before the startle test. Calves were brought to the arena in the same order each day, with order balanced across treatments. During habituation, no startle stimulus was delivered, but otherwise the same procedure followed on test days was applied.The startle test occurred between 1530 and 1800 h (Supplementary Video S1). The calf was transported from her home pen to the waiting pen in a cart (Caf-Cart, Raytec, Ephrata, PA). The test began when the gate providing access to the test pen was opened and ended after 10 min. The gate was closed behind the calf after she had entered so that the waiting pen was inaccessible during the test. Three observers were seated quietly 3.5 m away from the pen during the test, and were partially concealed behind a tree branch. One observer remotely controlled the speaker broadcasting the startle noise, while the other two observers were present to respond if a calf escaped from the arena (only one calf jumped out, on the first day of habituation, and was promptly escorted back into the pen). Calves showed no apparent responses to the observers and had no visual contact with other animals.As soon as the calf’s mouth was within a tongue’s reach of the teat, a 0.4 s, 105 ± 2 dB burst of white-noise was emitted through a wireless speaker (OT4200 Big Turtle Shell, Outdoor Tech, Laguna Hills, CA) mounted directly behind the bottle. The noise was created using an online signal generator23. We measured the sound level using a decibel meter (BAFX Products, Milwaukee, WI) held 30 cm in front of the bottle, approximating the distance of the calf’s ears to the source.Behavioural data collectionTests were recorded with a camcorder (HC-V180, Panasonic, Kadoma, Japan) positioned on a tripod approximately 3 m away from of the pen. One trained observer, blinded to the treatments, scored behaviours in all videos taken of the startle test and the third day of habituation (Table 1). Videos were analysed using BORIS (Behavioural Observation Research Interactive Software24). Intra-observer reliability was calculated using 12 randomly selected videos of the startle test (Intraclass correlation coeffcient ≥ 0.95).Table 1 Behavioural definitions used to evaluate calves’ responses in an arena test.Full size tableAccelerometers (Hobo Pendant G Acceleration Data Logger, Onset Computer Corporation, Bourne, MA) were used to assess the magnitude of the startle response. On habituation and test days, we fitted calves with a triaxial accelerometer set to record acceleration in the x-, y-, and z-axis every 0.05 s. The accelerometer was placed in a pouch, strapped around the right hind leg, and secured with Vet Wrap (Co-Flex, Andover Coated Products Inc., Salisbury, MA) while the calf was in the waiting pen of the arena, immediately before the gate to the test pen was opened. Data were downloaded using HOBOware Pro Software (Onset Computer Corporation, Bourne, MA). To calculate the magnitude of the startle response, we summed total acceleration in all 3 axes over the startle duration for that calf. Total acceleration was calculated as the square root of the sum of squared acceleration in each axis25. No startle response was recorded for one calf who did not approach the bottle on the test day.All calves were weighed the morning of the startle test (mean ± SD; Day 0: 56 ± 10 kg; Day 21: 55 ± 9 kg; Sham: 55 ± 11 kg).Wound healing and sensitivityWe measured sensitivity via mechanical nociceptive thresholds around the horn bud area 1 to 2 h after the startle test using a digital algometer fitted with a 4-mm-diameter round rubber tip (ProdPlus; TopCat Metrology Ltd., Little Downham, UK). The calf was restrained in the head device in her home pen and blindfolded to reduce responses to visual cues. We then applied an increasing amount of force to the edge of the disbudding wound, or intact horn bud for sham calves, as described previously13. The test ended when the calf moved her head or a maximum cut-off point of 10 N was reached. We repeated the test if a fly landed on the head, a loud noise occurred, or the calf urinated or defecated. If a test was interrupted 3 times, it was abandoned (0% of tests).Wound sensitivity was tested at the lateral and caudal edges of each wound or the equivalent location on sham calves. The order of test sites was: left lateral, left caudal, right caudal, and right lateral. To ensure force was applied at a consistent rate, personnel operating the algometer were trained and met a set of rigorous criteria before performing the tests13. We calculated the rate that force was applied in each test from video recordings (0.29 ± 0.10 N/s; 2% of videos missing). If force was increased at a rate  0.6 N/s or video was missing, the data were excluded (3% of tests). Due to the nature of the tests, the operator of the algometer was not blind to treatment.We took digital photographs of the wound with a DSLR camera (D5300; Nikon Corp., Tokyo, Japan) after sensitivity testing was completed. Photos were taken 15 cm from the wound. One person scored the photos for tissues present in the wound bed using a 0/1 scoring system13. Due to the clear differences in Day 0 and Day 21 wounds, the scorer was not blind to treatment.Statistical analysisDue to the presence of zeros in the data, we used zero-inflated beta regressions to assess the effect of treatment (Sham, Day 0, Day 21) on the proportion of time suckling on the third day of habituation and during the startle test. A zero-inflated beta regression is a mixture of two models: a beta model for estimating non-zero proportions and a logistic model for estimating the probability of zeroes26. This approach allowed us to infer treatment effects on both the occurrence and duration of suckling. General linear models were used to test the effect of treatment on the duration of the startle response and its magnitude as measured from the accelerometer data.We analyzed the effects of treatment on latency to approach the bottle and latency to return after startling using parametric survival regression models with a log-logistic distribution. Days on which the calf did not perform the behaviour within the allotted time (15 min for habituation, 10 min for startle test) were handled as right-censored data.We ran a general linear model to test the effect of treatment on wound sensitivity. A preliminary analysis indicated that there was no effect of side (left vs right) or location (caudal vs lateral) on wound sensitivity, so we averaged data for each calf into one score.Data were analysed in R, version 3.5.227. General linear models were fitted using the “lm” function in base R. 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Bioreactor operation and samplingA continuous-flow MBR made from Plexiglass with a working volume of 12 L was used for enrichment (Supplementary Fig. S1). The reactor was installed with a submerged hollow fiber ultrafiltration membrane module (0.02 μm pore size, Litree, China) with a total membrane surface area of 0.03 m2. A level control system was set up to prevent liquid overflowing. The reactor was fed with diluted real urine with Total Kjeldahl Nitrogen (TKN) concentration of 140–405 mg N L−1 (for detailed influent composition see Supplementary Table S1). Initially, the reactor was inoculated with activated sludge taken from the aeration tank of a municipal wastewater treatment plant (Tsinghua Campus Water Reuse). The pH was maintained at 6.0 ± 0.1 by adding 1 M NaOH to buffer acidification by ammonia oxidation. The airflow was controlled at 2 L min−1, leading to the dissolved oxygen (DO) concentration above 4 mg O2 L−1 as regularly measured by a DO probe (WTW Multi 3420). The airflow also served to wash the membrane and mix the liquid. The temperature was controlled at 22–25 °C. The initial hydraulic retention time (HRT) was 3 days and was decreased to 1.5 days on day 222. The sludge retention time (SRT) was infinite as no biomass was discharged.The MBR was operated for 490 days. During this period, influent and effluent samples (10 mL each) were collected 1–3 times per week and used to determine the concentrations of TKN, total nitrite nitrogen (TNN = NO2−-N + HNO2-N), and nitrate nitrogen, according to standard methods.19 Mixed liquid samples (25 mL) were also taken weekly to measure mixed-liquor suspended solids (MLSS) and mixed-liquor volatile suspended solids (MLVSS).19 Biomass samples (10 mL) were regularly taken for qPCR and microbial community analyses (see below).Batch testsIn order to test urea hydrolysis and subsequent nitrification in the enrichment culture, short-term incubations were performed in a cylindrical batch reactor (8 ×18.5 cm [d × h], made from Plexiglass). 150 mL biomass was sampled from the reactor and washed three times in 1 x PBS buffer to remove any remaining nitrogen source. Subsequently, the biomass was resuspended in a 400 mL growth medium, which contained urea (about 40 mg N L−1), NaHCO3 (120 mg L−1), and 2 mL Hunter’s trace elements stock. Dissolved oxygen was controlled above 4 mg O2 L−1. Biotic and abiotic controls were performed under identical conditions with NH4Cl (~40 mg N L−1) instead of urea. The pH in all batch assays was maintained at 6.0 ± 0.1 by adding 1 M HCl or NaOH. According to the microbial activities during long-term operation, each batch assay lasted 6 to 8 h, and samples (5 mL) were taken every 20 to 60 min. Biomass was removed by sterile syringe filter (0.45 μm pore size, JINTENG, China), and urea, ammonium, nitrite, and nitrate concentrations were determined as described above. All experiments were performed in triplicate.DNA extractionBiomass (2 mL) for DNA extraction was collected on days 0, 53, 98, 131, 161, 189, 210, 238, 266, 301, 321, 358, 378, 449, and 471. DNA was extracted using the FastDNA™ SPIN Kit for Soil (MP Biomedicals, CA, U.S.) according to the manufacturer’s protocols. DNA purity and concentration were examined using agarose gel electrophoresis and spectrophotometrically on a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA).16S rRNA gene amplicon sequencing and data analysisThe V4-V5 region of the 16 S rRNA gene was amplified using the universal primers 515F (5′-barcode-GTGCCAGCMGCCGCGG-3′) and 907 R (5′-CCGTCAATTCMTTTRAGTTT-3′).20 PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantified using the QuantiFluor™ -ST (Promega, USA). Amplicons were pooled in equimolar concentrations and sequenced using the Illumina MiSeq PE3000 sequencer as per the manufacturer’s protocol. Amplicon sequences were demultiplexed and quality filtered using QIIME (version 1.9.1).21 Reads 10 bp were assembled. UPARSE (version 7.0.1090 http://drive5.com/uparse/) was used to cluster operational units (OTUs) on a 97% similarity cut-off level, and UCHIME to identify and remove chimeric sequences. The taxonomy of each 16S rRNA gene sequence was assigned by the RDP Classifier algorithm (http://rdp.cme.msu.edu/) according to the SILVA (SSU132) 16S rRNA database using a confidence threshold of 70%.Quantification of various amoA by qPCRTo quantify the abundances of comammox Nitrospira, AOB and AOA in the bioreactor, qPCR targeting the functional marker gene amoA was performed on DNA extracted from the bioreactor at different time points. We used the specific primers Ntsp-amoA 162F/359R amplifying comammox Nitrospira clades A and clade B simultaneously,12 Arch-amoAF/amoAR targeting AOA amoA,22 and amoA-1F/amoA-2R for AOB amoA.23 Reactions were conducted on a Bori 9600plus fluorescence quantitative PCR instrument using previously reported thermal profiles (Supplementary Table S2). Triplicate PCR assays were performed the appropriately diluted samples (10–30 ng μL−1) and 10-fold serially diluted plasmid standards as described by Guo et al.24. Plasmid standards containing the different amoA variants were obtained by TA-cloning with subsequent plasmid DNA extraction using the Easy Pure Plasmid MiniPrep Kit (TransGen Biotech, China). Standard curves covered three to eight orders of magnitude with R2 greater than 0.999. The efficiency of qPCR was about 95%.Library construction and metagenomic sequencingThe extracted DNA was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. A paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments. Paired-end sequencing was performed on Illumina NovaSeq PE150 (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits according to the manufacturer’s instructions (www.illumina.com).Metagenomic assembly and genome binningRaw metagenomic sequencing reads (in PE150 mode) were trimmed and quality filtered with in-house Perl scripts as described previously.25 Briefly, duplicated reads caused by the PCR bias during the amplification step were dereplicated. Reads were eliminated if both paired-end reads contained >10% ambiguous bases (that is, “N”). Low-quality bases with phred values 2.5 kbp were retained for later analysis. Genome binning was conducted for each sample using sequencing depth and tetranucleotide frequency. To calculate coverage, high-quality reads from all samples were mapped to the contigs using BBMap v38.85 (http://sourceforge.net/projects/bbmap/) with minimal identity set to 90%. The generated bam files were sorted using samtools v1.3.1.27 Then, sequencing depth was calculated using the script “jgi_summarize_bam_contig_depths” in MetaBAT.28 Metagenome-assembled genomes (MAGs) were obtained in MetaBAT. MAG quality, including completeness, contamination, and heterogeneity, was estimated using CheckM v1.0.12.29 To optimize the MAGs, emergent self-organizing maps30 were used to visualize the bins, and contigs with abnormal coverage or discordant tetranucleotide frequencies were removed manually. Finally, all MAGs were reassembled using SPAdes with the following parameters: –careful –k 21,33,55,77,99,127. The reads used for reassembly were recruited by mapping all high-quality reads to each MAG using BBMap with the same parameter settings as described above.Functional annotation of metagenomic assemblies and metagenome-assembled genomesGene calling was conducted for the complete metagenomic assemblies and all retrieved MAGs using Prodigal v2.6.3.31 For the MAGs, predicted protein-coding sequences (CDSs) were subsequently aligned to a manually curated database containing amoCAB, hao, and nxrAB genes collected from public database using DIAMOND v0.7.9 (E-values < 1e−5 32) MAGs found to contain all these genes were labeled as comammox Nitrospira MAGs and kept for later analysis. Functional annotations were obtained by searching all CDSs in the complete metagenomic assemblies and the retrieved MAGs against the NCBI-nr, eggNOG, and KEGG databases using DIAMOND (E-values < 1e−5).Phylogenetic analysisPhylogenomic treeThe taxonomic assignment of the three identified comammox Nitrospira MAGs was determined using GTDB-tk v0.2.2.33 To reveal the phylogenetic placement of these MAGs within the Nitrospirae, 296 genomes from this phylum were downloaded from the NCBI-RefSeq database. The download genomes were dereplicated using dRep v2.3.234 (-con 10 -comp 80) to reduce the complexity and redundancy of the phylogenetic tree, which resulted in the removal of 166 genomes. In the remaining genomes, the three comammox Nitrospira MAGs and 25 genomes from phylum Thermotogae which were treated as outgroups, a set of 16 ribosomal proteins were identified using AMPHORA2.35 Each gene set was aligned separately using MUSCLE v3.8.31 with default parameters,36 and poorly aligned regions were filtered by TrimAl v1.4.rev22 (-gt 0.95 –cons 5037) The individual alignments of the 16 marker genes were concatenated, resulting in an alignment containing 118 species and 2665 amino acid positions. Subsequently, the best phylogenetic model LG + F + R8 was determined using ModelFinder38 integrated into IQ-tree v1.6.10.39 Finally, a phylogenetic tree was reconstructed using IQ-tree with the following options: -bb 1000 –alrt 1000. The generated tree in newick format was visualized by iTOL v3.40 amoA treeReference amoA sequences of AOB, AOA, and comammox Nitrospira were obtained from NCBI. Together with the amoA genes from the present study, all sequences were aligned and trimmed as described above. IQ-tree was used to generate the phylogenetic tree, with “LG + G4” determined as the best model.ureABC gene treeureABC gene sequences detected in this study were extracted and used to build a database using “hmmbuild” command in HMMER.41 ureABC gene sequences from genomes in NCBI-RefSeq database (downloaded on July 1st, 2019) were identified by searching against the built database using AMPHORA2. The same procedures as above were conducted to construct the phylogenetic tree of concatenated ureABC genes, except for the sequence collection step. To reduce the complexity of the phylogenetic tree, the alignment of concatenated ureABC genes was clustered using CD-HIT42 with the following parameters: -aS 1 -c 0.8 -g 1. Only representative sequences were kept for phylogeny reconstruction, which resulted in an alignment containing 858 sequences and 1263 amino acids positions. “LG + R10” was determined as the best model and used to build the phylogenetic tree. Regarding the Nitrospirae-specific ureABC gene tree, ureABC gene sequences were recruited from the genomes as described above, but without the sequence clustering step. The final Nitrospirae-specific phylogeny of ureABC genes was built on an alignment containing 62 sequences and 1015 amino acid positions with “LG + F + I + G4” as the best model. More