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Effects of water and nitrogen treatments on the yield of Isatis indigotica
As shown in Table 3, in the two-year experiment, both the water input and the nitrogen application rate had significant effects on the yield of Isatis indigotica.Table 3 Variance analysis of traits on the yield of Isatis indigotica.Full size tableAs shown in Fig. 4, with increasing water and nitrogen, the yield first increased and then decreased. The interaction between the water input and the nitrogen application rate reached a significant level (P  N1. At the levels of W1, W2, and W3, the yield of the N2 treatment increased by 5.3–7.9%, 6.5–6.9%, and 5.0–9.0% compared with those of the N3 treatment, respectively, and the yield of the N3 treatment increased by 1.4–1.9%, 1.5%-4.5%, and 1.7–3.5% compared with those of the N1 treatment, respectively. At the same nitrogen application level, the yield performance was W2  > W1  > W3. At the levels of N1, N2, and N3, the yield of the W2 treatment increased by 6.9–12.4%, 8.3–11.3%, and 6.8–13.5% compared with those of the W3 treatment, respectively, and the yield of the W3 treatment decreased by 1.6–3.9%, 1.5–1.6%, and 1.3–2.4% compared with those of the W1 treatment, respectively.Effects of the water and nitrogen treatments on the quality of Isatis indigotica
As shown in Table 4, in the two-year experiment, the water input and nitrogen application rate had significant impacts on the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharide in Isatis indigotica.Table 4 Variance analysis of traits the quality of Isatis indigotica.Full size tableAs shown in Fig. 5, The impacts decreased with increasing irrigation amount and nitrogen application rate. Compared with those in the W3N3 treatment, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharide in the W2N2 treatment increased by 4.5–5.9%, 2.7–3.1%, 5.2–6.0% and 1.8–2.1%, respectively. At the same irrigation level, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharides all decreased in the order N1  > N2  > N3. At the W2 level, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharide in the N1 treatment increased by 0.5–1.7%, 0.8–0.9%, 0.8–1.1% and 0.1–0.4%, respectively, compared with those in the N2 treatment. Compared with those in the N3 treatment, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharide in the N2 treatment increased by 1.9–2.1%, 1.5–2.2%, 2.1–2.2% and 0.6–1.1%, respectively.Figure 5The effects of the different treatments on the quality index of Isatis indigotica. The values shown are the mean ± SD, n = 3. Asterisks indicate a significant difference at the P ≤ 0.05 level.Full size imageAt the same nitrogen level, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharides all decreased in the order W1  > W2  > W3. At the N2 level, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharides in the W1 treatment increased by 1.5–2.0%, 1.8–2.1%, 3.0–3.1% and 0.4–0.9% compared with those in the W2 treatment, respectively. Compared with those in the W3 treatment, the contents of indigo, indirubin, (R,S)-epigoitrin and polysaccharides of the W2 treatment increased by 2.3–3.5%, 1.8–2.3%, 2.0–4.0% and 1.0–1.4%, respectively.Effects of the water and nitrogen treatments on the WUE of Isatis indigotica
As shown in Table 5, in the two-year experiment, the water input and nitrogen application rate had significant impacts on the WUE of Isatis indigotica (P  N3  > N1. At the W1, W2, and W3 levels, the WUE of the N2 treatment increased by 6.5–8.6%, 7.8–8.1%, and 7.4–10.4% compared with that of the N3 treatment, respectively, and the WUE of the N3 treatment increased by 2.9–3.1%, 3.9–6.0%, and 4.5–5.3% compared with that of the N1 treatment, respectively. Under the same nitrogen application rate level, the WUE performance was W1  > W2  > W3. At the N1, N2, and N3 levels, the WUE of the W1 treatment increased by 5.0–11.7%, 2.8–9.2%, and 2.0–10.9% compared with that of the W2 treatment, respectively, and the WUE of the W2 treatment increased by 24.2–29.5%, 24.3 -27.2%, and 23.5–30.3% compared with that of the W3 treatment, respectively.Effects of water and nitrogen treatments on NUE of Isatis indigotica
As shown in Table 6, in the two-year experiment, the water input and nitrogen application rate had significant impacts on the nitrogen fertilizer use efficiency (NUE) of Isatis indigotica (P  N2  > N3. At the levels of W1, W2, and W3, the NUE of the N1 treatment increased by 9.9–11.8%, 9.6–13.0%, and 6.3–11.6% compared with that of the N2 treatment, respectively, and the NUE of the N2 treatment increased by 31.0–37.6%, 28.8–29.2%, and 28.3–28.6% compared with that of the N3 treatment, respectively. At the same nitrogen application level, the NUE performance was W2  > W3  > W1. At the N1, N2, and N3 levels, the NUE of the W2 treatment increased by 5.7–6.1%, 2.5–4.8%, and 2.3–4.1% compared with that of the W3 treatment, respectively, and the NUE of the W3 treatment decreased by 3.4–8.0%, 6.9–8.2%, and 10.5–14.5% compared with that of the W1 treatment, respectively.Ethical guidelineThe authors confirm that relevant ethical guidelines were followed for plant usage.Land permit statementThe experimental land belongs to the Yimin Irrigation Experimental Station, in Minle County, Gansu Province, China. More

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Research model and softwareCLIMEX modelFAW growth and development are primarily related to climate conditions, especially temperature patterns17. The current study used CLIMEX (version 4)42, a semi-mechanistic niche modeling platform, to project FAW distribution in relation to climate. The model parameters that describe the species’ response to climate were overlaid onto FAW occurrence data and climate data to project the species’ potential global distribution. Briefly, the annual growth index (GI) was used to describe the potential for FAW population growth during favorable climatic conditions, while stress indices (SI: cold, wet, hot, and dry) and interaction stresses (SX: hot-dry, hot-wet, cold-dry, and cold-wet) (Table 1) were applied to describe the probability that FAW populations could survive unfavorable conditions. The Ecoclimatic index (EI) was derived from a combination of GI, SI, and SX indices to provide an overall annual index of climatic suitability on a scale of 0–10042. An EI value of 0 indicates that the location is not suitable for the long-term survival of the species, whereas an EI value of 100 indicates maximum climatic suitability comparable to conditions in incubators. EI values of more than 30 indicate the optimal climate for a species. In this study, the climatic suitability was classified into four arbitrary categories; unsuitable for EI = 0, marginal for 0  More

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Deer capture and samplingWe captured 100 mule deer (54 females, 46 males) during November 2018–February 2019, avoiding capture and sampling of juveniles. We attempted to distribute captures throughout the ~23 km2 study area described by Miller et al.5 to minimize spatial disparities in comparing contemporary and past data, and to assure marks were widely distributed for December ground counts to estimate deer abundance5,35,36. Field and sampling methods generally followed those used elsewhere5,31,37. Field procedures were reviewed and approved by the CPW Animal Care and Use Committee (file 14–2018).We pursued deer on foot and darted them opportunistically, delivering sedative combinations intramuscularly via projectile syringe. Premixed immobilization drug combinations included either nalbuphine (N; 0.9 mg/kg) or butorphanol (B; 0.5 mg/kg) combined with azaperone (A; 0.2 mg/kg) and medetomidine (M; 0.2 mg/kg)38, with standard total doses for respective combinations based on an estimated mass of 70 kg (average drug volume per animal was 1.3 ml NMA, 1.4 ml BAM). We collected rectal mucosa biopsies to determine CWD infection status37. We also collected whole blood and marked all deer with individually identifiable ear tags and some with telemetry (n = 51) or visual identification (n = 12) collars. Ages were estimated to the nearest year via tooth replacement and wear patterns39; observers used a pocket reference guide in the field to help assure consistency. To antagonize sedation upon completion of handling and sampling, each deer received 5 mg atipamezole/mg M administered, injected intramuscularly.Prion diagnosticsFormalin-fixed tissue biopsies were processed and analyzed by immunohistochemistry (IHC) at the Colorado State University Veterinary Diagnostic Laboratory (Fort Collins, Colorado USA; CSUVDL) for evidence of CWD-associated prion (PrPCWD) accumulations using monoclonal antibody F99/97.6.1 (VMRD Inc., Pullman, Washington, USA)40 and standard IHC methods24,37,41, except that the CSUVDL’s IHC staining machine (Leica Microsystems Inc., Buffalo Grove, Illinois, USA) was different from that used in earlier studies (Ventana Medical Systems, Oro Valley, Arizona, USA). Biopsies were evaluated microscopically and classified as positive (infected) or not detected (negative) based on PrPCWD presence or absence; the same pathologist (T. R. Spraker) read biopsies for both the current and prior5 studies.We included only data from deer with biopsies providing ≥3 lymphoid follicles in analyses involving infection status in order to maintain a relatively high (≥90%) probability of detecting infected individuals24. Two animals with low follicle counts that died shortly after capture were excepted by substituting postmortem IHC results. Limiting the acceptable follicle count excluded seven females (two 225SS, five 225SF) and two males (one 225SS, one 225SF) from some analyses. One male deer was 225FF and one female deer was missing a blood sample and thus not assigned to a PRNP gene group; these two individuals also were excluded from some analyses (e.g., Table 1).
PRNP genotypingWe used DNA extracted from whole blood buffy coat aliquots (n = 99) to screen for the presence of sequences at PRNP gene codon 225 that encode for serine (S) and/or phenylalanine (F) in the mature prion polypeptide, classifying individuals as 225SS, 225SF, or 225FF16,36,42. Methods generally followed those described by Jewell et al.16. Briefly, we extracted DNA using the DNeasy® blood and tissue kit (Qiagen, Valenica, California, USA). We amplified the complete open reading frame (ORF) plus 25 bp of 5′ flanking sequences and 53 bp of 3′ flanking sequences in the PRNP coding region using polymerase chain reaction (PCR). Purified DNA was combined in a 0.2 ml PCR tube containing a puReTag Ready-To-Go PCR bead (illustra™, GE Healthcare Bio-Sciences Corp, Piscataway, New Jersey, USA). Each PCR bead contained 2.5 units puReTag DNA polymerase, 10 mM Tris-HCI, 50 mM KCl, 1.5 mM MgCl2, 200 µM of each deoxynucleoside triphosphate, and stabilizers, including bovine serum albumin. For each PCR assay, 1 μL of each primer at 200 nM, 22 μL of RNase-free water and 1 μL of approximately 100 ng total genomic DNA was added for a final volume of 25 μL. Primers used for amplification were forward (MD582F, 5′-ACATGGGCATATGATGCTGACACC-3′) and reverse (MD1479RC, 5′-ACTACAGGGCTGCAGGTAGATACT-3′) described by Jewell et al.16. Reactions were thermal-cycled in a PTC 100 (MJ Research) at 94 C for 5 min and then 32 cycles of 94 C for 7 s, 62 C for 15 s, 72 C for 30 s and a final cycle of 72 C for 5 min, and kept at 4 C until inspected for successful amplification by agarose gel electrophoresis. As confirmed by LaCava et al.19, the MD582F and MD1479RC primers developed by Jewell et al.16 specifically amplify the functional PRNP gene ORF, thereby excluding confounding effects that could arise from the presence of a processed pseudogene that occurs in a majority of deer (Odocoileus spp.)42.We used EcoRI restriction digestion of the PCR-amplified PRNP region16—a validated assay targeting the singular polymorphism at codon 225 in mule deer—to screen all 99 samples for presence of S or F codons. Aliquots (10 μl) of completed PCR reactions were incubated with 10 U EcoRI (New England Biolabs) in a total volume of 12 μl containing 50 mM NaCl, 100 mM Tris/HCl, 10 mM MgCl2, 0.025% Triton X-100 (pH 7.5) at 37 C for 2–16 h followed by the addition of 2.5 μl 6× concentrate gel loading solution (Sigma- Aldrich) per sample, and the inspection of products by agarose gel electrophoresis for the presence of one 897bp-sized band for 225SS, two bands—one 897 bp and one 719 bp—for 225SF, or one 719 bp-sized band for 225FF. As noted by Jewell et al.16, occurrence of TTC (the F codon) at position 225 creates an EcoRI recognition DNA sequence and cleavage site GAATTC from codons 224–225, whereas TCC (the S codon) creates the sequence GAATCC, which is not cut by EcoRI. When incubated with EcoRI, PCR products with a TTC codon at position 225 yielded cleavage fragments of the predictable sizes listed16. Because no other sites within the PRNP ORF DNA sequence are potentially transformable to GAATTC with one base change, this represents a specific genotyping method for assessing the S225F polymorphism in mule deer16.To confirm findings from EcoRI screening, we examined sequences of the complete PRNP ORF from 20 samples that showed evidence of cleavage indicating 225*F and 6 samples without cleavage identified as 225SS. For DNA sequencing, we used primers 245 (5′-GGTGGTGACTGACTGTGTGTTGCTTGA-3′), 12 (5′-TGGTGGTGACTGTGTGTTGCTTGA-3′) and 3FL1 (5′-GATTAAGAAGATAATGAAAACAGGAAGG-3′; Integrated DNA Technologies). Sanger sequencing was done on purified PCR product by Eurofins Genomics (Louisville, Kentucky, USA). Sequence chromatograms were viewed and DNA sequence alignments and comparisons were made using the MAFFT multiple sequence alignment program v7.450 module, software platform v2020.2.3 of Geneious Prime. Sequencing confirmed the presence of coding for F in all samples identified as 225*F by EcoRI digestion, as well as the absence of such coding in samples identified by EcoRI digestion as 225SS. Moreover, presence of AGC at codon 138 in all sequenced samples reconfirmed that the primers we used had amplified the functional PRNP gene42.Statistics and reproducibilityFor analyses, we tabulated IHC-positive and -negative results to estimate apparent prevalence of prion infection. We also tabulated the number of individuals assigned to PRNP genotypes and to age groupings as described. Age groupings were selected based on relevance to CWD epidemiology in mule deer1,5,8,12,16,17,18,20,24,31,37. Assuming a ~2-year disease course5,8,17 and relative scarcity of end-stage disease in 225SS deer More