Ecology

Seeds collection and germination
We collected T. sebifera seeds by hand from populations in both the introduced (US—16 populations in total) and native (China—14 populations in total) ranges (for details see Table S1). At each population, we haphazardly selected 5–10 trees, and harvested thousands of seeds from each tree. In the laboratory, we removed the waxy coats around these seeds by hand after immersing them in a mixture of water and laundry detergent (10 g/L) for 24 h [29]. Then, we rinsed, surface sterilized (10% bleach), and dried them. In order to improve germination, we put these seeds in wet sand and stored them in the refrigerator (4 °C) for at least 30 days. In spring, we sowed these seeds in greenhouse trays (50 holes/tray) which were filled with sterilized (autoclave at 121 °C for 30 min) commercial potting soil, and then kept them in an open-sided greenhouse at Henan University in Kaifeng, Henan, China (34°49′13′′ N, 114°18′18′′ E) or unheated greenhouse at Rice University, Houston, TX USA (29°43′08′′ N 95°24′11′′ W). After seeds germinated and seedlings reached the 4 true leaf stage, we selected similar size seedlings to conduct the following experiments.
Common garden experiment—differences in AM fungal colonization and plant growth
To investigate the differences in AM fungal colonization and growth between plants from introduced (US) and native populations (CH), we carried out a common garden experiment at Henan University. We collected soil in a corn field, which includes most common AM fungal species based on previous reports [33, 34]. It was a sandy soil with total nitrogen and total phosphorus of 1.9 g/kg (DW) and 0.6 g/kg (DW), respectively, and pH of 7.68. We removed surface litter before collecting topsoil (10–15 cm depth) and then combined equal parts of soil and fine sand in 132 pots (21 cm × 16 cm, ~3 kg of soil mix each) after they were passed through a 1-cm mesh screen. We planted seedlings from 22 populations (12 native and 10 introduced populations, 6 seedlings of each population, Table S1) individually in these prepared pots and placed them in the open-sided greenhouse. We protected them from herbivores with nylon mesh (16 openings/cm) cages during the experiment. After 60 and 90 days, we harvested 3 seedlings from each population as 3 reps each time and carefully washed their whole roots from the soil. We collected ~30 fresh fine roots ( >1 cm/segment) from each plant root to measure AM fungal colonization. In brief, we cleared (in 10% KOH), bleached, acidified, and stained (trypan blue) these samples then slide mounted 30 one cm long pieces of fine root for each plant [7]. AM fungal colonization was determined by the gridline intersect method with 300 intersection points per plant [35]. We dried and weighed the roots and shoots.
Collection of root exudates and flavonoids analysis for root exudates
Our previous study found higher concentrations of flavonoids but lower concentrations of tannins in roots of introduced populations of T. sebifera than in native populations [17] with quercetin and quercitrin being the main flavonoids [28, 30]. In our pilot experiment, we only detected quercetin and quercitrin in root exudates but no other flavonoids. Therefore, in this study we focused on quercetin and quercitrin in root exudates and their functions. We determined their amounts in root exudates from native (China) and introduced (US) populations at Henan University. We filled 132 glass beakers (1000 ml) with Hoagland’s solution [36] and covered the opening with a foam board with a hole in its center. We took 6 seedlings from each of 22 populations (12 native, 10 introduced, Table S1) and carefully washed the soil from their roots with tap water, then transplanted them individually into the beakers (1 seedling per beaker) and fixed them with a sponge. Because of mortality, only 80 plants of 17 populations (9 native, 8 introduced) survived until exudate collection. The odds of a plant dying did not depend on population origin (F1,20 = 3.7, P = 0.0679) or population (Z = 1.3, P = 0.0937). We checked these glass beakers and filled them with Hoagland’s solution every day.
After these plants grew for 57 or 87 days in an open-sided greenhouse with a typical temperature range of 18 °C (night) to 28 °C (day) and 13–14 h of natural daylight, we put DI water into these beakers instead of Hoagland’s solution to minimize the effects of environments on root exudates. Three days later (i.e., at 60 and 90 days) these plants were harvested to obtain their dry root mass. The root exudates were dried at 40 °C under vacuum by rotary evaporators. Then we extracted the chemicals from these concentrates in 3 ml of methanol solution with 0.4% phosphoric acid water (48:52, v:v) and filtered them through 0.22 μm hydrophobic membranes. The concentrations of quercetin and quercitrin were assessed by high-performance liquid chromatography [30]. In brief, 20 μl of extract was injected into an HPLC with a ZORBAX Eclipse C18 column (4.6 × 250 mm, 5 μm; Agilent, Santa Clara, CA, USA) with the following flow: 1.0 mL min−1 with a 100% methanol (B) and 0.4% phosphoric acid in water (A) as the mobile phase. The gradient was as follows: 0–10 min 52:48 (A:B); 10–24 min 48:52 (A:B). UV absorbance spectra were recorded at 254 nm. The concentrations of flavonoid compounds were calculated and standardized by peak areas of standards of known concentrations.
Root exudate addition experiment—effects of different populations on AM fungal colonization
In order to investigate the role that root exudates play in the interactions between AM fungi and plants, we conducted an experiment in which exudates were collected from plants in liquid (donor) and applied to the soils of other plants (target). The exudate donor plants were grown in 1080 (two venues: 540 seedlings at Rice University and 540 seedlings at Henan University) containers, each with 1000 ml of Hoagland’s solution, that each had a foam board top with a hole and a bottom drain tube that could be regulated. At each venue, we washed the soil from ~500 sets of plants (US = 465, China = 504) from native (8 populations for venue US and 7 populations for venue CH) or introduced (13 populations for venue US and 12 populations for venue CH) populations and secured them (3 plants per container) in the containers using sponges (details in Table S1). The remaining containers were left as plant-free controls. We started the application experiment after 7 days.
For exudate target plants, we collected the soil from different sites in the introduced or native ranges (See Table S1). At each site, we collected soil under the canopy of a T. sebifera tree (Home soil) and that more than 3 meters away from the canopy of a T. sebifera tree (Away soil). We collected the topsoil to a depth of 15 cm after removing the surface litter, air dried them, and screened them (1 cm mesh). These soils were mixed with vermiculite (1:2 volume). Then we used these mixes to fill 1080 pots (15 cm × 12 cm; 540/venue). Each pot in China received a mixed soil from a site in China and each pot in US received soil from a single small area within a site in the US. We transplanted a seedling from a native (12 populations for venue US and 3 populations for venue CH, See Table S1) or introduced (13 populations for venue US and 5 populations for venue CH, See the Table S1) population into each pot (270 of each per venue). We randomly assigned a target plant to each set of donor plants or water only controls.
Every 4 days we changed the Hoagland’s solution to DI water for 3 days to collect root exudates from donor plants. Then we applied this water solution from a donor set to its target plant. After 70 days, we harvested the target seedlings, kept a fine root sample for AM fungal colonization determination, then dried and weighed leaves, stems, and roots.
Chemical addition experiment—quercetin and quercitrin effects on AM fungal colonization
We transplanted 391 seedlings from 8 native populations (CH) and 9 introduced populations (US) into 391 pots with field soil (1.3 kg/pot) in nylon mesh cages at Henan University. To test the effect of quercetin and quercitrin on AM fungal colonization, we prepared solution of quercetin or quercitrin in acetone (10 mg/mL) (acetone did not affect AM fungal colonization based on our preliminary experiment). Then these solutions were diluted in water to 2 concentrations (1 mg/L and 10 mg/L) based on the result of chemical analyses of root exudates and the 0.1% of acetone in water as control. We watered 15 ml of solution (5 reps per population) or water (3 reps per population) around the base of seedling stems every 3 days (16 times in total). Four plants died (3 in quercitrin application treatment, 1 in quercetin application treatment). After 70 days, we collected seedlings by cutting at ground level and collected fine roots to test AM fungal colonization.
Activated carbon experiment—AM fungal colonization with inactivated chemicals
In order to verify the chemicals in root exudates play a key role in the relationship between AM fungi and plant roots, we conducted an experiment at Henan University with activated carbon (AC) addition to block bioactivity of root exudate chemicals. We filled plastic pots in mesh cages at Henan University with either 1.3 kg of field soil (N = 78) or field soils amended with activated carbon (N = 78, Sinopharm Chemical Reagent Co., Ltd, Beijing, China) added as 1:500 v:v. We transplanted seedlings from 13 populations (6 native and 7 introduced, Table S1) into the pots with 6 seedlings for each population. Eighteen seedlings died during this experiment (12 seedlings from AC treatment, 6 seedlings from control). After 70 days, we harvested plants and used a fine root sample to determine AM fungal colonization.
Field survey of AM fungal assemblages
We collected rhizosphere soil from 3 sites in China (Dawu, Hubei, 31°28′N, 114°16′E; Wuhan, Hubei, 30°32′N, 114°25′E; Guilin, Guangxi, 25°04′N, 110°18′E) for AM fungal species identification via high-throughput sequencing. At each of these sites, we selected 3 T. sebifera trees per site and dug the soil close to the tree trunk until its root branch was found. We collected soils from 3 roots per plant. We removed the bulk soil from these roots by shaking, and then collected the soil remaining on these roots using brushes (1 new brush per tree). The rhizosphere soils on the roots from same tree were mixed fully. About 5 g of fresh rhizosphere soil from one tree was collected and stored in dry ice and ultra-low temperature freezer (−80 °C) until they were used to test the AM fungi abundance based on high-throughput sequencing [37, 38].
For DNA extraction, microbial DNA was extracted from the prepared samples (0.25 g soil per sample) using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the manufacturer’s protocols. The DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA), and DNA quality was checked by 1% agarose gel electrophoresis [39].
For the PCR amplification, nested PCR was conducted to amplify the AM fungi 18S rRNA. The primer pairs AML1 (5′-ATCAACTTTCGATGGTAGGATAGA-3′) and AML1 (5′-GAACCCAAACACTTTGGTTTCC-3′) were used in the first run. The primer pairs AMV4.5NF (5′-AAGCTCGTAGTTGAATTTCG-3′) and AMDGR (5′-CCCAACTATCCCTATTAATCAT-3′) were used in the second run in the thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were conducted using the program according to Xiao et al. [39].
For each sample, purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw fastq files were quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) the reads were truncated at any site receiving an average quality score More

Experimental design
The Winchmore Irrigation Research Station is in the centre of the Canterbury plains, the largest area of flat land in New Zealand (43.787° S, 171.795° E; Fig. 1). It is at an altitude of 160 m above sea level, a mean annual temperature of 12 °C, and has an annual rainfall of 745 mm (range 491–949 mm)20. The soil is a Lismore stony silt loam classified as an Orthic Brown soil in the New Zealand soil classification and as an Udic Ustochrept in USDA soil classification21. Flood irrigation, known locally as border-check/dyke irrigation, was installed at the site in 1947. However, the two long-term trials, hereafter known as the fertiliser and irrigation trials, were established in 1952 and 1949, respectively.
Fig. 1

Location of Winchmore within the Canterbury region (coloured green) and the layout of the long-term fertiliser and irrigation trials over time.

Full size image

Full details of the setup of the fertiliser and irrigation trials between 1949–1951, including the political rationale for the trial, its statistical design, cultivation dates, sowing rates of perennial ryegrass (Lolium spp) and white clover (Trifolium repens) and initial fertiliser and irrigation treatments are available elsewhere20.
The fertiliser trial has 20 border check irrigation bays divided into five treatments each with four replicates set out in a randomised block design (Fig. 1). From 1952/53 to 1957/1958 treatments were: nil (no P applied), 188, 376 (annually and split P applications), and 564 kg SPP ha−1. All P applications occurred annually in autumn except for the 376 kg SSP ha−1 treatment which had two treatments divided into an annual autumn application and split applications in between autumn and spring. From 1958/59 to 1979–80 the nil and 188 and 376 (split autumn and spring application) SSP treatments were unaltered, while P applications were stopped to the annual 376 and 564 SSP treatments. In 1972, 4.4 t/ha of lime (caclium carbonate) was applied to all treatments22. From 1980 onwards the nil, and 188 SSP treatments and the 376 SSP treatment, now receiving winter fertiliser applications, were joined by a treatment applying 250 SSP ha−1 in winter to the previous 376 SSP treatment and a Sechura rock phosphate treatment applying 22 kg P ha−1 in winter to the former 576 SSP treatment.
Each irrigation bay was fenced off, 0.09 ha in size and grazed by separate mobs of sheep at 6, 11, and 17 stock units (with one stock unit equivalent to one ewe at 54 kg live-weight) per replicate for the nil, 188 SSP, and 376 SSP treatments, respectively. This separation prevented carry-over of dung P and other nutrients and contaminants between treatments. No grazing occurred in winter. Flood irrigation was applied when soil moisture content (w w−1) fell below 15% (0–100 mm depth). This occurred on-average 4.3 times per year.
The irrigation trial had 24 irrigation bays (each 0.09 ha in size) which had lime applied to the whole trial in 1948 (5 t ha−1) and 1965 (1.9 t ha−1) to maintain soil pH at 5.5–6.0. From 1951 to 1954 treatments were SSP applied at 250 kg ha−1 in autumn annually and either four replicates of dryland, or five replicates of irrigation applied at one, two, three, six-weekly intervals or at three-weekly intervals in alternate seasons. From 1953/54 to 1956/57 the weekly and two-weekly treatments were replaced by irrigation when soil moisture in the top 100-mm of soil reach 50 and 0% available soil moisture (asm), respectively. In 1958 the irrigation trial was cultivated with a rotary hoe and grubber, 140 kg SSP ha−1 applied and the site re-sown in ryegrass and white clover. From 1958/59–2007 the site had the same blanket application of SSP and four replicates of dryland, while a completely randomised design was used to impose five replicates of four treatments (Fig. 1) that looked at irrigation applied when soil moisture in the top 100-mm of soil reach 10, 15 and 20% (equivalent to 50% asm with 0% asm being wilting point) and irrigation on a 21-day interval. The need for irrigation to the irrigation and fertiliser trials was informed by soil moisture measured weekly by technical staff using a mixture of gravimetric analyses (1950–1985), neutron probe (1985–1990) and time-domain reflectometer (1990-onwards). Irrigation was applied at a rate of 100 mm per event20.
Except for winter, when no grazing occurred, each treatment was rotationally grazed by a separate flock of sheep with 6 and 18 stock units per replicate for the dryland and 20% v/v treatments, respectively.
The irrigation trial finished in October 2007 although the P fertiliser regime continued. All irrigated treatments shifted to the same three weekly schedule as the long-term Fertiliser trial. The dryland treatment remained unirrigated. The Winchmore farm was converted into a commercial irrigated farm operation and sold in 2018. The fertiliser trial was also sold but with a covenant ensuring it continues to operate as per normal except that irrigation from 2018 onwards is now applied by spray irrigation with the aim of ensuring soil moisture is maintained above 90% of field capacity. Since January 2019 there are daily soil moisture meter records from a moisture meter installed into one of the control plots. Soil moisture, rainfall and irrigation are recorded.
The production of the Winchmore trials data records23 involved a three-step process (Fig. 2).
Fig. 2

Flowchart of the steps involved in sampling, analysis, collation and curation and data analysis and processing of the databases from the Winchmore Trials. Note that blue and orange boxes are sub tasks associated with each step and resulting outputs, respectively.

Full size image

Step 1: Soil and pasture sampling
Pasture production was measured from two exclusion cages (3.25 m long × 0.6 m wide) per plot24. Areas within each cage were trimmed to 25 mm above ground level and left for a standard grazing interval for that time of year. Following grazing a lawnmower was used to harvest a 0.40 m wide strip in the middle of each enclosure to 25 mm above ground level. The wet weight was determined, and a sub-sample taken to determine dry matter content with a separate sample manually dissected into grass, clover and weeds. All surplus mown herbage was returned to the plot. Approximately 9–10 cuts were made annually. A composite soil sample of 10 cores (2.5 cm diameter and 7.5 cm deep) was collected from each plot. These were collected four times annually (July, prior to fertiliser application, and October, January and April), using established best practices24,25. In 2009 soil samples were also collected from the 0–75, 75–150, 150–250, 250–500, 500–750, and 750–1000 mm depths on both trials17. During 2018, prior to cultivation, soil on the unirrigated, 10 and 20% soil moisture treatments of the irrigation trial were sampled at 0–150, 150–250, 250–500, 500–750, 750–1000, 1000–1500, and 1500–2000 mm depths. The top 250 mm of these samplings were collected by hand using an auger, but deeper depths were excavated via a mechanical digger. Representative sub-samples were taken from each depth. Annual samplings were crushed, dried and sieved More

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Composition of chemical constituents and bacterial communities in AGS and feces indicates separate, unique odor profiles
The gas chromatography–mass spectrometry analyses revealed that AGS volatiles of wild and captive pandas were comprised of a multicomponent blend of 30–50 chemical compounds, including fatty acids, aldehydes, ketones, aliphatic ethers, amides, aromatics, alcohols, steroids and squalene (Fig. 2a and Supplementary Table S2). These compounds are typical components of chemosignals across species due to their volatility, detectability and other characteristics facilitating chemoreception [3, 26, 32]. By contrast, feces contained mostly fatty acid ethyl ester, and a small number and quantity of fatty acids, amides, steroids and indole (Fig. 2b and Supplementary Table S3). Our results show that the relative abundance of steroids, aldehydes and fatty acids were remarkably higher in AGS than in feces (Fig. 3a), and the number of chemical components of aldehydes, fatty acids, and ketones in AGS was also significantly higher than found in feces (Fig. 3b). These results indicate that the chemical constituents of AGS are much better suited for chemosignaling than those from feces.
Fig. 2: Representative ion chromatograms of samples in giant pandas.

a Anogenital gland secretions (AGS). b Feces.

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Fig. 3: Differences in chemical compounds of anogenital gland secretions (AGS) and feces in giant pandas, and the differences in microbial communities, KEGG and contribution bacteria for lipid metabolism.

a A heat map of the mean relative abundance of the chemical compounds. b A heat map of the number chemical components. Differences in the microbial communities as a function of providence (captive/wild) and source (feces/AGS) at the c phylum and d genus level. e PCoA clustering results of samples from different groups. f Hierarchical clustering analysis of the samples, clearly indicating two branches for AGS and fecal samples. g Six differentially represented pathways in lipid metabolism and the Linear discriminant analysis (LDA) score. h Prevalence of enzymes involved in lipid metabolism as a function of phylum and family in AGS of giant pandas. i The contribution of different bacteria at genus level to lipid metabolism. WPF: wild panda feces, CPF: captive panda feces, WPAG: wild panda AG, CPAG: captive panda AG.

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The composition of bacterial communities in AGS and feces was markedly different at the phylum (Fig. 3c) and genus levels (Fig. 3d), based on taxonomic classifications of predicted gene sequences. Principal Co-ordinates Analysis (PCoA) (Fig. 3e) and hierarchical clustering analyses (Fig. 3f) revealed cluster patterns based on provenance (captive/wild) and sample type (AGS/fecal). Notably, the microbiota composition of AGS from different individuals or living environments was more similar than were AGS and fecal samples from the same individuals. Actinobacteria (X2 = 26.33, P  More

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We used high-throughput qPCR to screen for 58 infective agents in four Atlantic salmon farm cohorts from British Columbia throughout their production cycles. We measured presence and copy number for target genetic sequences, characteristic of specific viral, bacterial, and eukaryotic agents, including several recently discovered viruses30,31, known or suspected to cause disease in salmon. These agents displayed various temporal patterns of prevalence and intensity, with several displaying elevated levels in dead and dying fish.
The data and analyses we have presented provide a unique look into the epidemiology of farmed salmon populations, and wildlife/livestock diseases generally. No past studies have had access to multiple farmed-salmon cohorts, throughout their production cycles, with the capacity to molecularly screen for a large suite of infectious agents. Other work has reported agent data for dead-sampled fish collected in BC as part of Fisheries and Oceans Canada’s farm audit program40, but such analyses lack the time-series nature of the results we have presented. To our knowledge, no other studies have provided such detailed, comprehensive information for infective agents in domestic or wild populations over time. This study, therefore, presents a substantial step toward effectively monitoring shared wildlife/livestock diseases, made possible by cutting-edge technology, as predicted previously22.
While our findings offer specific insight to salmon farmers, aquaculture managers, and those concerned with the disease ecology of sympatric wild salmon, we caution that results remain correlative, and relevant patterns (e.g. apparent mortality signatures) require further investigation. Unfortunately, a lack of regular testing of dead and dying fish (collection was opportunistic, at farms’ discretion) resulted in potential for associated patterns to be obscured. Due to this potential bias in the sampling design, we are unable to draw conclusions related to farm-level mortality rates, but several agents showed patterns of note, including elevated levels in dead and dying fish.
Agent patterns
Perhaps the clearest single-agent pattern—the elevated load of T. maritimum in dead and dying fish (Fig. 3B)—matches generally accepted patterns in aquaculture. Induced tenacibaculosis can be responsible for substantial on-farm mortality worldwide41, and mouthrot resulting from T. maritimum in the east Pacific causes substantial losses42. In our study, mouthrot was noted during veterinarians’ sample processing for cohorts one, three, and four in the months after ocean entry. We note that elevated levels in dead and dying fish could represent the bacterium’s acknowledged role as an opportunistic pathogen41, rather than a direct cause of mortality. We also note the positive correlations between T. maritimum load and that of a number of different agents (Fig. 5), consistent with the view that T. maritimum may facilitate co-infections43. Given its high overall prevalence in fish (Table 3), secondary factors—such as co-infections—might exacerbate infection with T. maritimum, playing a role in mortality.
K. thyrsites intensity was elevated in dead and dying fish for cohorts three and four, around the time they were transferred to their final marine locations (Fig. S8). In both cases, the cohorts finished their production cycles in farms on the central east coast of Vancouver Island (Fig. 1), a region in which the risk of K. thyrsites infection is acknowledged to be high44. This myxozoan parasite is economically important due to post-mortem myoliquefaction seen in infected fish, but it is not considered a pathogen45, and it is unclear why higher gene-copy levels would be observed in dead/dying fish. K. thyrsites may merely replicate faster in stressed fish (in this case due to transport). Our surveillance of pathogens did not include skeletal muscle tissue, where K. thyrsites spores develop, but it did include heart, which can be infected by the parasite46. We note that K. thyrsites was correlated with PRV, with both agents known to infect muscle tissue (although red blood cells are the primary infective tissue for PRV). Follow-up histopathological investigation may provide some insight into K. thyrsites distribution and any associations with pathology or patterns of co-infection.
PRV, which is the causative agent of Heart and Skeletal Muscle Inflammation (HSMI)47 and has recently generated controversy in BC28,29,48, shows several patterns of note. PRV prevalence increased to near ubiquity over time (Fig. 2D), concurrent with an increase, peak, slight decline, and then stabilisation of intensity (Fig. 3B). Although our perspective is limited to sampled fish, with a noted potential for bias, the observed PRV trends were consistent across all four cohorts, and the intensity patterns are consistent with previously reported dissemination, peak replication, and long-term persistence phases of the virus within hosts29,48. Past findings suggest that PRV may induce an antiviral response in hosts that can protect them against certain co-infections49,50. Perhaps counter to the generality of this claim, PRV and ASCV exhibited the strongest load correlation out of any we observed across our data set (Fig. 4). ASCV was originally isolated from salmon with HSMI, and was initially thought to play a role in the disease51. Other work has found no relationship between ASCV and PRV infections or HSMI52. In the case of a related baitfish calicivirus, however, there is evidence that viral co-infection is linked to disease manifestation53, so further work is needed to tease these relationships apart. In general, dead and dying Atlantic salmon in our study did not show elevated prevalence or intensity of PRV, except shortly after ocean entry in cohort one (Figs. 2D, 3B). This mortality signature corresponds to the onset of lesions diagnostic of HSMI in cohort one, which subsequently spread to affect the majority of that farm population for most of a year29.
The gill chlamydia bacterium, C. Syngnamydia salmonis, showed a consistent trend towards elevated prevalence in dead and dying salmon (Fig. 2B). Observed intensity was low, however, often averaging approximately a single copy (Fig. S15). Sequencing has validated past detections of this agent on the Fluidigm BioMark™, and has also revealed SNP diversity within the primer-binding region, resulting in potential underdetection (Miller et al. unpublished). Given a putative mortality signature and the lack of prior epidemiological study of this recently discovered agent54,55, we would suggest further work on C. Syngnamydia salmonis.
Ephemeral mortality signatures appeared for several agents. F. psychrophilum was clearly elevated in dead and dying fish in-hatchery, although we only had access to two hatchery cohorts and cannot draw general conclusions. Intensity of both the ASCV and CTV-2 were elevated in sampled dead and dying fish from at least one cohort shortly after ocean entry (Figs. 3A, S2). Both viruses were also present in-hatchery. The previously reported Cutthroat trout virus appears to be apathogenic56 in trout, and has been detected in Atlantic salmon57. Little is known about the novel variant for which we screened, although in situ hybridisation has revealed that infection can be systemic and extensive in the brain (Mordecai et al. 2020). As for the ASCV, associated pathology was found to be likely due to PRV contamination51. Extremely limited information about these two viruses, paired with our findings, warrants further investigation (e.g. with histopathology and in situ hybridisation) to determine if either virus is linked with pathology. As both these viruses were detected in Chinook salmon (Mordecai et al. 2020), and considering their high prevalence in Atlantic salmon farms, the potential risk they pose to wild Pacific salmon populations should be a priority for future research.
Infectious agent levels overall, as measured by relative infectious burden, showed a clear trend in smolts coming out of freshwater hatcheries for cohorts three and four. There, infectious burden was much higher (in one case 10 000 times higher) in dead and dying fish than in live-sampled fish. While the effect dissipated once fish entered the marine environment, it is clear that hatchery fish are dying with—or of—elevated levels of infection. The patterns we observed likely reflect the transition from freshwater to saltwater, with a coincident shift in infective-agent communities58. Smoltification has also been associated with immune depression59, and elevated infectious burden around the time of ocean entry may reflect this. Where we had dead/dying hatchery samples, however, infectious burden was elevated weeks before ocean entry, hinting at the potential for problems in-hatchery.
Across all agents, we observed apparent coinfection signals that clearly differed from random chance (Figs. 5, S21). We point out, however, that due to the longitudinal nature of our study and cursory investigation of agent correlations, the correlation results almost certainly indicate shared temporal trends that may or may not indicate underlying interactions. For example, Candidatus Branchiomonas cysticola and Flavobacterium psychrophilum were positively correlated with each other, but negatively correlated with a number of other agents. This could be due to both agents being common in-hatchery but not in the marine environment (Figs. S3, S6), counter to many other agents’ patterns. Alternatively, the correlation could be due to a biological relationship, perhaps in relation to gill health. We intend to follow up on a number of correlational patterns.
Agent idiosyncrasies
Several agents showed unexpected patterns, or patterns that may be connected to their particular biology.
The putative Narna-like virus, a recently discovered agent31, showed elevated prevalence in dead and dying fish (Fig. S9). This pattern was mainly due to over-representation in dead-sampled fish, as we detected the agent in 13.2% of dead fish, 1.6% of moribund fish, and 0.4% of live-sampled fish in saltwater. Given that Narnaviridae, of which the putative Narna-like virus is a member, is thought mainly to infect fungi60, this virus may be associated with a fungal decomposer. This is speculative, however, and recent genomic evidence from across taxa suggests that the Narnaviridae may be much more widespread than previously thought61.
Counter to the common trend, P. pseudobranchicola tended to be less common in dead and dying fish than in live-sampled fish (Fig. 2C), with dead fish, in particular, tending to exhibit the lowest levels (results not shown). P. pseudobranchicola primarily infects the pseudobranch62, a structure near the gills involved in oxygenating blood in the eye. Infection also occurs in tissue collected for this study, especially gill63, and we speculate that loads in dead fish could be reduced due to myxospore release or degradation of delicate gill tissue after host death. Given that we did not sample the pseudobranch, it is likely that our data underestimates the load of this organism.
The sampling environments (freshwater or marine) of several detections were unexpected. In particular, we detected K. thyrsites and T. maritimum (Fig. 3C) in freshwater hatcheries, although these agents are considered marine species64,65. It is possible that these hatcheries introduced saltwater in the weeks before ocean transfer, to prepare smolts for release. We also detected F. psychrophilum, considered a freshwater bacterium66, in marine net pens (Fig. 2A). The bacterium is known to survive in brackish water67, however, and this is not the first time it has been detected in a marine setting40,68.
Broader connections
Not all infective agents cause disease, and even agents that do can be present long before—or long after—clinical symptoms. Our work presents only a piece of the puzzle in what is a multifaceted, complex scenario of shared wildlife/livestock disease in salmon aquaculture. The controversy surrounding PRV in BC, as an example, illustrates this complexity. While conventional lab challenges using PRV from BC sources have failed to reproduce in BC fish the extent of HSMI lesions observed on Norwegian farms48,69, work related to our study has been able to identify and shed light on HSMI, and related jaundice/anemia in Chinook salmon, in BC salmon farms28,29. While we saw a putative mortality signature in one cohort during this study, the normal course of PRV infection was not always associated with mortality (e.g. Figs. 2D, 3B). More work will be required to elucidate the nuances of PRV infection, factors that induce associated disease, and possible resultant mortality. A fruitful place to start would be to carry out sampling and diagnostics of dead and dying fish in farms and pens experiencing elevated mortality.
Although we have shown putative mortality signatures for several infective agents in farmed Atlantic salmon, these are not necessarily the agents that pose the greatest risk to wild salmon. For one thing, a given agent need not produce the same effects in different species28,70. For another, contact between populations may not coincide with infection maxima. Depending on when farm smolts enter the marine environment, for example, PRV could be at low prevalence in the spring, when a number of wild Pacific salmon species migrate as juveniles15. Other times of year would be more relevant for interactions with other wild species, and there is much scope for transfer between farmed and wild environments. In addressing shared wildlife/livestock disease, we need to consider both wildlife and livestock as populations that serve as potential reservoirs of disease agents, and are susceptible to outbreaks71. In this context, surveillance and monitoring are essential facets of disease management23, providing raw material to develop understanding of disease and build effective management strategies. Parallel work is monitoring wild populations for the same agents we have investigated here, with the prospect of cross-referencing patterns and impacts72,73,74.
The ubiquity of infectious agents on the farms leads naturally to discussion of potential control strategies, which present a variety of challenges in aquaculture. Vaccination has proven successful at times, but the salmon aquaculture industry has a somewhat chequered history with uptake, since vaccination can affect host growth, and thus the bottom line13. In addition, vaccines have only been developed for a handful of agents. Reducing translocations can be an effective control strategy on land20,78, but transmissive properties of the marine environment and highly mobile marine carrier hosts pose challenges to isolating host populations geographically (Krkosek 2015). Our findings provide circumstantial evidence that some agents (e.g. K. thyrsites) respond to translocations. The fact that two of our four focal cohorts moved substantial distances throughout their respective marine production may be cause for concern, considering the infective-agent populations we have shown those cohorts to have harboured. In general, aquaculture-associated disease and related management decisions have a history of generating political controversy75. Infective-agent monitoring and analyses are critical for designing, implementing, and evaluating effective disease-control measures, and for bridging divides in debate surrounding aquaculture.
With respect to the aquaculture industry, the tools we employed in this study may prove useful for disease management and fish health. We have shown that for many agents, patterns of infection in dead and dying fish mirror those in live fish. By integrating high-throughput infectious-agent screening with existing monitoring of dead fish, farm vets and managers could access a wealth of otherwise unavailable or costly information. Combining such results with strategic sacrificial sampling of live fish during mortality peaks could allow additional insight into which agents may be driving mortality. Protecting the ‘herd’ (and its wild neighbours) may justify such mortal sampling. Furthermore, in other ongoing work, we have seen that much infective-agent information is accessible via nonlethal gill biopsy, which also enables high-throughput screening for gene expression patterns associated with various patterns of stress76 and disease77. Used appropriately, such a combination of tools could be very powerful.
Disease monitoring is never complete, and detection always lags behind pathogen spread78, but new technologies—such as those we employed here – can facilitate efficient, lower-cost surveillance and monitoring. Surveillance for existing pathogens and identification of previously unknown pathogens is part of the integrative approach required to understand and control existing and emerging infectious diseases22. Here, we have further demonstrated the utility of high-throughput, modern genetic techniques for monitoring known infective agents and for generating information about previously under-studied agents26,29,30,31,37,38. Further work will target the risk of transfer between wild and farmed hosts and prioritize threats to salmon, farmed and wild. More

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