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ESP sample processing
The ESP operated autonomously, needing only power, communications and fluid connections through its waterproof pressure housing (Fig. 1). Prior to sample initiation, the ESP was purged completely with nitrogen to reduce oxidative reactions (i.e., DNA degradation) from occurring. At the initiation of sampling, a puck (Fig. 1A cutout) loaded with filter material was placed within a clamp. Valves open to the outside allowed a syringe to sequentially pull water through the puck. Once the target volume was filtered, or the filter was loaded with biomass (i.e., ‘clogged’), filtering stopped and excess water was cleared. Five mL’s of RNAlater preservative was then added to the puck, soaking the filter for 10 min before the excess was evacuated and the puck was returned to storage. Preserved pucks were stored at the ESP temperature, which were similar to ambient air temperatures. The upper limit on the amount of time that an ESP device can operate in the field before DNA quality on a puck is comprised is not known but is at least 21 days10. A constant humidity kept the pucks moist, allowing for easy filter removal once the instrument was recovered.
Figure 1

The ESP is an electro-mechanical robot that can autonomously filter and preserve samples. (A) About the size of a 50-gal barrel, the ESP carries 132 ‘pucks’ (inset), each designed to hold 25 mm filters. (B) The ESP installed in a USGS streamgage station. (C) Streamgage station showing tubing run (white pipe) that contained pump and tubing to deliver stream water to the ESP. The ESP communicated via cell phone, and was powered during the deployment via either line power or portable solar arrays. Photo credits: U.S. Geological Survey.

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To get water to the ESP, we designed an external sampling module from which the ESP drew water11. The sampling module was self-draining, and fed by a submersible pump (WSP-12 V-2 M, Waterra USA Inc., Bellingham, W, USA) installed approximately 0.5 to 2 m below the river water line at each deployment site. To reduce possible carry-over contamination, the sampling pumps, tubing and external sampling modules were flushed with river water for 10 min prior to every sample collection. The sampling port of the ESP itself was cleaned with 10% bleach and a 10% tween-20 solution between samples. At the end of each ESP deployment, pucks were manually removed and filters were aseptically recovered into 2.0 mL screw cap centrifuge tubes and stored at − 80 ºC until molecular analyses were performed.
Field deployments
We performed initial ESP feasibility studies in Yellowstone National Park (USA; Fig. 2) in September 2017. Here, our goal was to determine if the ESP could be used to sample DNA of the waterborne protozoa, Naegleria spp., from a freshwater river where these organisms had previously been detected using standard techniques12. We filled 60-L sterilized carboys with water from the confluence of the Boiling and Gardner rivers. Carboys were transported to a lab at Montana State University (Bozeman, Montana) and connected to ESP samplers via tubing and syringe pumps. Water was passed through each filter (5-µm Diapore filters) until the filter became clogged; six samples were filtered.
Figure 2

Map of ESP water sampling locations. The inset map shows the location of the Upper Yellowstone River and Upper Snake River in the United States. The larger map shows the sample site locations (filled red circles) on each river relative to Yellowstone National Park and Grand Teton National Park (outlined in green).

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We then integrated the ESPs into two USGS streamgages on the Yellowstone River in 2018 and one USGS streamgage on the Snake River in 2019, (Fig. 1B,C) where we tested for DNA of the fish pathogen, Tetracapsuloides bryosalmonae, the causative agent of salmonid fish Proliferative Kidney Disease (PKD). On the Yellowstone River, we installed ESPs at the streamgage near the upstream and downstream extents of a recent PKD outbreak13, USGS 06191500 Yellowstone River at Corwin Springs MT and USGS 06192500 Yellowstone River near Livingston MT, described below as Corwin Springs and Carters Bridge, respectively (Fig. 2). On the Snake River, we installed one ESP at the streamgage 1.5 km downstream of Palisades Reservoir near the upstream extent of a recent PKD outbreak, USGS 13032500 Snake River near Irwin ID. The ESP pucks were loaded with 1.2-µm cellulose nitrate filters. We ran two negative controls (1 L of molecular grade water) through the ESP prior to and at the conclusion of deployment to assess for contamination.
Yellowstone River
The ESPs were programmed to collect 1-L samples every 12 h, from Jul 24 to Aug 26 2018, and every 3 h from Aug 27 to Sep 7 2018. The average (± 1 SE) volume filtered per sample was 639 (± 11) mL, indicating that most filters clogged prior to reaching the 1-L target volume. Filter samples were collected at ambient air temperatures ranging from 9.6 to 35.8 °C ((overline{x})  = 18.9) at Carter’s Bridge and 8.3–29.0 °C ((overline{x})  = 17.1) at Corwin Springs. We compared T. bryosalmonae ESP detections to those from manually collected grab samples from shore (6, 250-mL samples per site filtered through 1.2-µm cellulose nitrate filters) collected at weekly frequencies for the entire length of the ESP deployments and at daily frequencies between Aug 27 and Aug 30. Thus, ESP and manual eDNA samples collected at different temporal intervals (3 h, 12 h or weekly) allowed us to evaluate the added value of higher frequency sampling.
We also evaluated the utility of automated high frequency sampling to detect a new invasion by introducing novel DNA of Scomber japonicas (mackerel fish) 100 m upstream of each Yellowstone River streamgage. On Aug 27, we introduced 3 kg of canned S. japonicas 100 m upstream of the water sampling inlet for each ESP. S. japonicas was blended with water, frozen and then placed within metal-wire minnow traps and anchored to the river’s bottom with cement pavers. The ESPs were programmed to sample every 3 h from Aug 27 to Sep 7. Manual grab samples (600 mL) were collected 10 m (n = 3), 100 m (n = 6), and 400 m (n = 3) downstream of the S. japonicas in order to test that S. japonicas DNA was transported downstream past the water sampling inlet of each ESP. Manual grab samples were collected immediately prior to S. japonicas introductions, 3 h post-introduction and then every 24 h for 3 days.
Snake River
The ESPs were programmed to collect 2-L samples every 12 h from Jul 17 to Sep 09 and then every 4 h from Sep 10 to Oct 1, 2019. Manually collected grab samples (three, 2-L samples filtered through 1.5-µm glass fiber filters) and negative field controls (1, 2-L sample of deionized water filtered through 1.5-µm glass fiber filters) were collected every 2 weeks following methods in Sepulveda et al.7. Filter samples were collected at ambient air temperatures ranging from 3.9 to 30.2 °C ((overline{x})  = 20.6). To broaden our taxonomic assessment, we tested these samples for T. bryosalmonae DNA, and also for kokanee salmon (Oncorhynchus nerka) and dreissenid mussel (Dreissena spp.) DNA. O. nerka only occur upstream in Palisades Reservoir and at such low abundances that they are not captured by resource managers in annual population surveys7. Dreissenid mussels have not yet been observed, but are the principal focus of aquatic invasive species monitoring programs in this region7.
Molecular analyses
Filters were removed from the pucks and then shipped frozen to the USGS Upper Midwest Environmental Science Center (LaCrosse, Wisconsin) for DNA extraction and quantitative PCR analyses. Filters were handled and stored in a dedicated room that is physically separated from rooms where high-quantity DNA extraction and PCR product or high-quality DNA is handled. We used the FastDNA SPIN kit for soil to extract DNA on samples from the Boiling River-Gardiner River confluence, following modifications described in Barnhart et al.14. To extract DNA from Yellowstone River and Snake River samples, we used the Investigator Lyse & Spin Basket Kit (Qiagen, Hilden, Germany) in concert with the gMax Mini genomic DNA kit (IBI Scientific), following manufacturer’s instructions, and eluted in 200 µL of buffer. Samples were extracted as site specific batches and one extraction control was collected per batch. We used previously published assays, limits of detection and methods therein for analyses of Naegleria spp.12, T. bryosalmonae13, S. japonicas15, O. nerka7, and Dreissena spp.16 (Table 1).
Table 1 Primers and probes used in this study.
Full size table

We analyzed all samples in four replicate 25 µL reactions containing 2 µL of template DNA, 1 × Perfecta Toughmix (Quantabio), 400 nM forward and reverse primers, and 100 nM probe. Each plate contained 10 no-template PCR controls (one for each sample) using 2 µL of molecular grade water as the template as well as a standard curve with two replicates of 20,000 and 2,000 copy standards and four replicates of 200 and 20 copy standards. The standards were prepared with synthetic gBlocks (Integrated DNA Technologies) containing the amplicon sequences for each assay. Each sample was also analyzed in three replicates with 200 copies of synthetic gBlock spiked in to check for PCR inhibition. Any sample that indicated less than an average of 60 to 70 copies of targeted DNA in these triplicate samples was considered inhibited. Field and extraction negative controls were analyzed as regular samples. No negative controls amplified.
Analyses
Samples were scored as positive when one or more PCR replicates amplified for the target DNA. We used McNemar’s Exact Test to compare binary qPCR data (detection/non-detection) of T. bryosalmonae and O. nerka DNA between ESP and manually collected samples in the Yellowstone and Snake rivers. More

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Wheat cultivars and growing conditions
In this study, pot and field experiments with the shade-tolerant cultivar Henong825 and the shade-sensitive cultivar Kenong9204 were performed. These two winter wheat cultivars were identified with different degrees of shade tolerance by our previous study17. Both cultivars are released by Hebei Province, China, which are the most widely planted wheat cultivars in North China Plain. The parental combination of Henong825 and Kenong9204 is Linyuan95-3091/Shi4185, SA502/6021, respectively. Henong825 is characterized by strong lodging resistance. Kenong9204 is characterized by suitable for moderate water and fertilizer. Two field experiments were conducted during the 2016–2017 and 2017–2018 wheat-growing seasons in the Luancheng agro-ecosystem experimental station of the Chinese Academy of Sciences, Hebei Province (37° 53′ N and 114° 41′ E; elevation at 50 m). The climate characterizing of the study region is summer monsoon. The mean temperature, total precipitation, and solar radiation in both the winter wheat-growing seasons are shown in Table 5. The soil used in the experiments was loam containing 21.41 g kg−1 organic matter, 109.55 mg kg−1 alkaline nitrogen (N), 1.44 g kg−1 total N, 15.58 mg kg−1 available phosphorus (P), and 220 mg kg−1 rapidly available potassium (K). In both seasons, soils were fertilized with urea (N, 46%) and complete fertilizer (N–P, 21–54%) at 300 kg ha−1 and 375 kg ha−1. Seeds were sown by hand on October 6, 2016 and October 17, 2017, then the seedlings emerged 1 week later. In 2017 growing season, the YM stage was on April 15, and anthesis stage was on May 1 in both cultivars. In 2018 growing season, the YM stage was on April 16, and anthesis stage was on May 2 in both cultivars. The seedling density was 166 m−2, which is the norm in this region.
Table 5 The monthly mean temperature (°C), total precipitation (mm), and solar radiation (MJ m−2 day−1) during the two growing seasons of winter wheat in 2016–2017 and 2017–2018.
Full size table

Experimental design
This study was a combination of field experiment and pot experiment to investigate the effect of different shading intensity and duration during YM stage on grain components and photosynthetic characteristics. Pot experiment was supplement to field experiment.
Field experiments
The experiments were arranged in a randomized split-split plot design with three replicates. The main plots were split into three subplots subjected to one of three shading intensities: 100% (CK, control), 40% (S1), and 10% (S2) of natural light. Each subplot was split into four sub-subplots, which were randomly allocated to one of four durations: 1 day (D1), 3 days (D3), 5 days (D5), and 7 days (D7) during the YM stage. The shading treatments were conducted in these periods and replicated three times. Each plot size was 6 m long and 2 m wide, with 40 rows. There were 72 plots. Different degrees of artificial shade were provided by using black polyethylene screens horizontally installed at a height of 2 m above the ground.
Determination of YM stage
The YM stage roughly corresponds to Zadok’s scale from Z37 (main stem with flag leaf is visible) to Z39 (flag leaf ligule is noticeable). According to previous researches of YM stage, the estimated measurement of the YM stage was based on the auricle distance (AD, the distance between the auricle of the flag leaf and the auricle of the penultimate leaf) of main stem43,44. In order to keep the relationship between the occurrence of YM and AD unchanged, the field management practices, adequate irrigation was the same in two growing-seasons. Moreover, for each experiment, at the onset of appearance of the flag leaf of the main stem, 30 anthers of ten main stem spike of wheat were randomly sampled to establish the timing of YM stage initiation1. The correlation of the AD with the development of the YM stage in the florets of the two cultivars was measured and observed using microscope (Fig. 9). The cultivar Henong825 reached the YM stage at 1–2 cm, whereas Kenong9204 reached the YM stage at − 1 to 0 cm. To capture the YM stage in the shading condition, the plants were subjected to shading stress ahead of the YM stage occurrence. When more than 50% of the plants in each plot reached − 2 cm in Henong825 and − 4 cm in Kenong9204, the main stem of the plants was tagged, and shading stress was applied in each plot. Each experimental plot for Henong825 and Kenong9204 was independently subjected to shading stress on April 15, 2017 and April 16, 2018. When the shading stress treatments ended, the shade screens were removed and were exposed to natural light until they matured. Air temperature, light intensity, and relative humidity above the canopy were recorded using a portable weather station (ECA-YW0501; Beijing, China) during the shading period. Light spectral was measured using a portable geographic spectrometer (PSR + 3500, USA). The irradiance of spectral wavelength ranging from 350 to 2,500 nm was measured. The proportions of blue light (B/T), green light (G/T), red light (R/T), far-red light (FR/T), and red/far red (R/FR) were calculated according to their irradiance at 400–500 nm, 500–600 nm, 600–700 nm, and 700–800 nm, respectively. Following the local field management practices, adequate irrigation was conducted three times during the overwinter, jointing, and anthesis stages of the wheat-growing season. Weeds, fungal diseases, and insect pests were controlled through spraying of conventional herbicides, fungicides, and insecticides, correspondingly.
Figure 9

The relationship between anther development and shading period in two wheat cultivars.

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Pot experiments
The pot experiments were conducted in a temperature-controlled glasshouse. Vernalized seedlings of the two wheat cultivars were transplanted to pots (45 cm in length, 28.5 cm in width and 20 cm in height; 18 plants in each pot; three pots for each treatment group) containing a mixture of vermiculite and nutritional soil (1:1). All wheat seedlings were grown at a day temperature of 25 °C, night temperature of 15 °C, and light intensity of 800 μmol m−2 s−1. When the AD of the main stems of Henong825 and Kenong9204 cultivars were approximately − 2 cm and − 4 cm, respectively, the main stem of the plants was tagged, and shading stress was applied in each treatment. Shading treatments groups were the different shading intensities and shading durations previously mentioned. The shading condition in glasshouse was simulated with black polyethylene screen to keep up with the experimental methods in the field. After shading stress, the shading nets were removed, until the crops matured.
Sampling and measurements
Photosynthetic rate, stomatal conductance, intercellular carbon dioxide, and chlorophyll fluorescence parameters
In field experiments, three randomly selected flag leaves on the tagged main stems of plants in each plot were analyzed to determine Pn, Gs, Ci, and chlorophyll fluorescence. For each shading treatment group, Pn, Gs, and Ci were measured using an LI-6400XT portable system (LI-COR Biosciences, Nebraska, USA), and the chamber of which was equipped with a red/blue LED light source (LI6400-02B) before the shading stress was removed. Before measurement, the machine was preheated for 30 min, and checked, adjusted to zero, calibrated according to the instructions. Moreover, the light intensity in measured chamber was equivalent of shading treatment conditions. The flow rates was set at 500 μmol s−1, The temperature in chamber was set 25 °C. The CO2 concentration was set to 400 μmol mol−1, which was provided by carbon dioxide cylinders to maintain a stable CO2 environment. The chlorophyll fluorescence of flag leaves on the tagged main stems of plants were measured using a modulate chlorophyll fluorescence imaging system (Imaging-PAM; Hansatech, UK) in each plot. The primary light energy conversion efficiency of PSII (Fv/Fm) and actual photochemical quantum efficiency (YII) were measured after 30 min of dark adaptation. The saturation irradiance (PARsat) and maximum electron transport (Jmax) of flag leaves in each treatment were calculated using a modified rectangular hyperbola. On the day next to shading removal, the Pn of three flag leaves from each replicate plot were measured.
Chlorophyll content
For glasshouse pot experiments, nine flag leaves (three leaves were randomly selected per pot from three pots in each treatment group) tagged main stems of plant were selected prior to the removal of shading. The flag leaves were then sliced following the removal of the main vein. After the sliced fresh leaves were weighed to 0.1 g, the chlorophyll content of leaves was extracted with 80% acetone for 48 h and analyzed through micro-determination (Thermo Varioskan Flash, USA). The absorbance of chlorophyll a (chl a) and chlorophyll b (chl b) was read at 663 and 646 nm, respectively (Thermo Varioskan Flash, USA), and the chlorophyll contents were calculated according to following equations: chl a (mg/g) = (12.7 × A663 nm–2.69 × A646 nm)/(100 × M); and chl b (mg/g) = (22.9 × A646 nm–4.68 × A663 nm)/(100 × M) where A663 and A646 are absorption levels at 663 and 646 nm, respectively; M is leaf fresh weight. The total chlorophyll (chl a + chl b) values were calculated by chl a and chl b values.
Leaf anatomy and surface characteristics
The approximately 2-mm2 leaf sections in D7 treatments and one day after recovery were harvested from the center of three flag leaves on the tagged main stems of plants using a scalpel and were rapidly fixed in electron microscope fixation fluid at 4 °C overnight. Stomatal apertures and chloroplast ultrastructure were observed by Servicebio (Wuhan) using a scanning electron microscope (SU8100; Hitachi) and a transmission electron microscope (HT7700; Hitachi). Simultaneously, the fully expanded flag leaves collected from plants in each treatment were fixed with FAA solution and embedded in paraffin to measure the leaf anatomical structure. The embedded wax block were sectioned to a thickness of 8 μm, then following dewaxing in environmental transparent solution and rehydration in a series of graded alcohol solutions. Finally, the tissue samples were stained with safranin and fast green, observed under a Leica DM6 microscope (Leica, Germany), and the respective images were obtained.
Grain yield, yield components, and aboveground biomass
At harvest in the field experiments during both growing seasons, 60 tagged plants per replicate were randomly sampled to determine grain yield components. The harvested plants were naturally dried to a grain water content of approximately 11%. Each tagged plant was then threshed using a single plant threshing machine to determine the grain number and grain yield needed for the estimation of the average grain weight. In addition, 30 tagged winter wheat plants were uprooted randomly and gradually by hand from each plot. Each plant was cut from the root and was dried at 80 °C. Aboveground biomass was measured using a precision digital balance (model BSA3202S; Sartorius, Germany) with a precision of 0.01 g.
Statistical analysis
The experimental data for grain yield, yield components, biomass and chlorophyll fluorescence parameters were analyzed using a general linear model procedure (GLM) in SPSS 22.0 for a split-split plot design. The significant differences among treatment mean values were determined by the least significance difference analysis (LSD, P  More