Grass Carp

Based on the published draft genome of grass carp [32 (link)], we cloned the sequences of CPT Iα1b and CPT Iα2a promoters. Genomic DNA was extracted from grass carp tail fins using a commercial kit (Omega, Norcross, GA, USA). For amplification of the CPT Iα1b and CPT Iα2a promoter sequences, specific primers with overlapping sequence were designed and listed in Table 1. For the generation of the luciferase reporter constructs, the PCR product and pGl3-Basic vectors (Promega, Madison, WI, USA) were purified and digested using corresponding endonucleases, and then products were ligated using ClonExpress™ II One Step Cloning Kit (Vazyme, Piscataway, NJ, USA). According to the distance from its TSS, the plasmids were named as pGl3-CPTIα1b-2695 and pGl3-CPTIα2a-2632, respectively. Plasmids pGl3-CPTIα1b-2276, pGl3-CPTIα1b-1716, pGl3-CPTIα1b-1073, pGl3-CPTIα1b-581, pGl3-CPTIα1b-86, pGl3-CPTIα2a-2041, pGl3-CPTIα2a-1646, pGl3-CPTIα2a-1304, pGl3-CPTIα2a-1165, pGl3-CPTIα2a-848, pGl3-CPTIα2a-428 and pGl3-CPTIα2a-97, which contained unidirectional deletions of the promoter regions, were generated with the Erase-a-Base system (Promega) using templates of pGl3-CPTIα1b-2695 and pGl3-CPTIα2a-2632, respectively. The PCR reactions were performed using TaKaRa PrimeSTAR^® HS DNA Polymerase kit (TaKaRa) as mentioned above. All plasmids were sequenced in a commercial company (Tsingke).

pCMV-CMV-GFP was employed as original plasmid (28 (link)) for constructing the following expression plasmids: LGP2-Flag, RIG-I-Flag, MDA5-Flag, RIG-I-HA, RIG-I-CARD-HA, RIG-I-Helicase-HA, RIG-I-RD-HA, MDA5-HA, MDA5-CARD-HA, MDA5-Helicase-HA, MDA5-RD-HA, RIG-I-CARD-Flag, and MDA5-CARD-Flag. The ORFs or the domains of the relevant genes were amplified from grass carp spleen tissue cDNA and then inserted behind the first CMV promoter. The Flag or HA tag was introduced by the reverse primer. The primers were listed in Table S1 in Supplementary Material. To construct the luciferase reporter vectors of grass carp IPS-1, MITA, IRF3, IRF7, IFN1, IFN2, IFN3, IFN4, IFNγ1, IFNγ2, NF-κB1, and NF-κB2, the 5′-flanking fragments of these genes were obtained from the grass carp genome (37 (link)). The core promoter regions were predicted by WWW Promoter Scan,¹ GPMiner,² and Promoter 2.0 Prediction Server.³ To verify the promoter activities, the predicted promoters of the related genes were introduced to the pCMV-GFP vector by replacing the CMV promoter (38 (link)). The primers were shown in Table S1 in Supplementary Material. Then, these vectors were transfected into CIK cells by FuGENE 6 Transfection Reagent (Promega), respectively. The promoter activity was reflected by promoting green fluorescent protein expression, observed under a fluorescence microscope (Nikon). The promoter activities of RIG-I, MDA5, and MITA were verified in the previous studies (39 (link)–41 (link)). For dual-luciferase reporter assay, the valid promoters were cloned into pGL3-basic luciferase reporter vector (Promega), respectively. For transient transfection, CIK or FHM cells were plated in 24-well plates, 6-well plates, or 10 cm² dishes with 70–90% confluency. Approximately 24 h later, transfection was performed with FuGENE 6 Transfection Reagent (Promega) according to the manufacturer’s instructions. LGP2 stable transfected cell line was obtained by G418 selection as previously reported (38 (link)). It is worth noting that the vector (pCMV-CMV-EGFP) used for protein overexpression in the present study contains two CMV promoters, which promote the expressions of target protein and EGFP, respectively, and the later is used to monitor the transfection efficiency. Hence, we employed empty vector-transfected cells rather than normal cells as control in the present study, which can make a better demonstration of LGP2 functions in RLR signaling pathways, not EGFP or other components in the vector skeleton. To assess the influence of empty vector on dual luciferase reporter assay, transcription level, and protein expression, we compared the promoter activities, mRNA expressions, and protein levels between empty vector-transfected cells and normal cells, and the results demonstrated that empty vector has no significant influence on the promoter activity, mRNA level, and protein synthesis (Figures S1 and S2 in Supplementary Material; Figure 3E).

Liver, spleen and kidney tissues from diseased Chinese giant salamanders were cut into pieces and homogenized in phosphate buffered saline (PBS) with antibiotics (100 IU penicillin mL^-1, 100 μg streptomycin mL^-1). Extracts were stored overnight at -20 °C, thawed, clarified by centrifugation at 2000 × g and filtered through a sterile 0.45 μm filter (Millipore, Billerica, MA, USA). The filtered supernatant was used as the original viral isolate for infecting cell lines and stored at -80 °C.
Epithelioma papulosum cyprini (EPC), Fathead minnow (FHM), Grass carp fins (GCF), Grass carp ovary (GCO), Bluegill fry (BF-2) and Chinook salmon embryo (CHSE) cell lines maintained in our laboratory were used for virus isolation and sensitivity tests. The above tissue homogenates were inoculated onto confluent monolayers of these cells maintained in TC 199 medium with 5% fetal bovine serum at 20 °C. When advanced cytopathic effects were observed, the cell culture supernatants were harvested and used as virus stocks for virus passages and characterization. Virus titers were measured on the basis of 50% tissue culture infective dose (TCID₅₀)/mL as described previously [14 (link),28 (link)].

We used a generalized additive model (GAM) with a Poisson link function to model the proportion of individuals in the telemetered population migrating upstream each day during the spawning season, and included the total number of fish with tracking data available each day as an offset term. The model was fit using the ‘gam’ function in the ‘mgcv’ package [47 ] in R [42 ]. We included daily covariates for temperature, stage, discharge, and an interaction term between stage and discharge because there was not always a linear relationship between the two in 2019 as the reservoir filled and backflow was observed further upstream. Daily covariate values represent the mean stream gauge reading for each day, and all covariates were scaled and centered. Given the long monitoring period encompassed a wide range of water temperatures through spring and summer (April–August) and considering grass carp spawning likely occurs within a relatively narrow temperature range (18–24°C; Jones et al. 2017), water temperature was included in the GAM as a smooth term to account for the nonlinear effect we anticipated temperature to have on fish movement. We modeled linear relationships between migration and all other covariates to avoid overfitting and because we had no a priori assumptions of non-linearity. Parametric coefficients and smooth terms with P values < 0.05 were considered significant. Additionally, we used Lin’s concordance correlation coefficient (CCC) to assess the fit of the predicted proportion from the GAM to the observed portion of fish migrating [48 ].
We used a logistic regression model to determine if ploidy (categorical), total length at stocking, and wet weight at stocking affected the probability of a fish engaging in migratory behavior. For this analysis we incorporated fish with tracking data during the spawning season (April–August). Fish were considered migratory if they were observed making ≥ 1 upstream migratory movement during this time. We scaled and centered total length and wet weight. We considered covariates significant if the 95% confidence interval of the parameter estimate did not encompass 0.

We recorded grass carp locations in rkm upstream from the dam to characterize large-scale upstream movements. We associated locations in wider parts of the reservoir or adjacent coves to the closest point to the river channel using the rkm at that point. This method assumes the shortest distance a fish could have traveled, although the distance could be longer if a fish traveled in a nonlinear path. Distance between locations was calculated as the shortest path a fish could take through the reservoir and tributaries upstream.
We cannot confirm grass carp that made large-scale upstream movements spawned or were intent on spawning, but seasonal upstream migrations to areas suitable for spawning has been observed in grass carp [39 (link)] and other invasive carp species [40 (link)]. As such, we assumed that upstream movements during the spring/summer seasons were associated with spawning activity in our analyses. Time between detections was irregular, making it difficult to statistically compare movement behavior among individual fish. Therefore, we scaled individual fish movement to regular 12-h time steps and estimated the distance moved (rkm) using the ‘redisltraj’ function in the package ‘adehabitatLT’ [41 (link)] in R [42 ]. We used 12-h steps because consecutive detections at different locations were rarely < 12-h and intervals greater than this may miss quick movements associated with the start of a potential migratory run. Following Acre et al. [43 (link)], we identified individual 12-h steps as part of a migration when distance moved was ≥ 85^th percentile of all observed distances moved over a 12-h period. Each movement step was then binomially categorized as part of a migration (1) or not (0).
We quantified river conditions based on data from the Osage River gage at rkm 116.7 (USGS gage 06918250). Temperature (°C), discharge (m³/s), and stage (m) are measured every 15 minutes at this location. We are aware that conditions were unlikely to be exactly homogeneous throughout the study area; however, as it was not feasible to measure the entire river, we make the necessary simplifying assumption that measurements at this location were representative of general conditions of discharge and temperature. River conditions along movement paths were based on gauge readings at the time of each 12-h interpolated location. The importance of these variables for successful spawning of grass carp is well documented, including ideal temperatures for optimal ripening and development of eggs post fertilization as well as higher velocities and turbulence associated with increased discharge and river stage to keep eggs suspended prior to hatching [36 (link), 44 (link)]. In addition, these variables have been associated with grass carp spawning movements in previous studies [45 , 46 ]. Although other environmental variables may play a role in grass carp movements (i.e., turbidity, food availability), it was not feasible to quantify them on a large spatial scale.

Truman Reservoir is the largest reservoir in Missouri and was formed by the damming of the Osage River near Warsaw, Missouri in 1979 [34 ] by the U.S Army Corps of Engineers. Truman Reservoir has a surface area of > 22,500 ha and four major tributaries, the Osage River, Pomme de Terre River, South Grand River, and Tebo Creek (Fig 1). Another major tributary, the Sac River, empties into the Osage River just above the reservoir at river kilometer 80 (rkm, river kilometers upstream from Truman Dam). Grass carp have likely been present since the formation of the reservoir via inundation of ponds in the watershed that were stocked with grass carp for vegetation control, and potentially through natural recruitment or continued escapement from ponds in the watershed where grass carp are stocked for vegetation control [35 , 36 (link)]. Because the reservoir was filled before triploid grass carp production technology was developed, and the production and stocking of diploid grass carp remains legal in Missouri [30 ], all escapees from ponds inundated by the initial filling of the reservoir would have been fertile, diploid fish. Many fish that later escaped from ponds in the watershed would also have been fertile. The Missouri Department of Conservation, which manages the Truman Reservoir fishery, does not stock grass carp into the reservoir [20 (link)]. With the discovery of successful spawning through the collection of fertilized eggs on four Truman Reservoir tributaries [20 (link)], it became evident that natural recruitment has likely increased the population in the system.