We moved 64 larvae to a 30 L container with aerated, carbon-filtered water (water from at ~20°C for three days to acclimate to the experimental temperature. Afterwards, all larvae were staged (Gosner, 1960), weighed, and divided into two treatments: 1) uninfected controls (N = 24) and 2) ranavirus infected (N = 40). The ranavirus used for inoculation was a frog-virus 3-like (FV3) isolate cultured in baby hamster kidney cells (BHK-21) to a concentration of 1.3 x 108 pfu ml-1. Briefly, individual larvae were placed in 750 mL plastic containers with 200 mL of water containing an LD¬100 concentration of 104.25 pfu ml-1 FV3. After 24 hours, 400 mL of water was added along with crushed alfalfa pellets. Uninfected controls underwent a similar protocol except they were only exposed to water. 2.3 Experimental Protocol At 2- and 4-days post infection (dpi), the CTmax of 12 randomly selected uninfected and infected larvae was recorded. Individual larvae were placed in a 125 mL flask with 75 mL of water, submerged in a hot water bath (Isotemp 220, Fischer Scientific), and given 5 minutes to acclimate. Starting temperature of CTmax assays averaged 18.8°C with a heating rate of 0.6°C minute-1. Beginning at ~34°C, larvae were prodded with a spatula every minute until they failed to respond to the stimulus, and water temperature was recorded which represented the larval CTmax. Flasks were then placed in a water bath at ~19°C to facilitate recovery. Individuals that failed to recover in < 5 minutes were removed from all analyses. Infected larvae were returned to their respective container and uninfected larvae were euthanized via snap freezing with -80°C EtOH. After each CTmax assay, all materials were sterilized in 10% bleach for > 10 minutes. To determine how the CTmax assay affected ranaviral loads, infected larvae that underwent the CTmax assay at 2 dpi were staged, weighed, and euthanized via snap freezing at 3 dpi. Another eight infected larvae whose CTmax was not measured were randomly selected and euthanized to represent the non-heat shocked group. This process was repeated for the larvae assayed at 4 dpi. All larvae were stored at -80°C. 2.4 Ranavirus DNA Extraction and Quantification The liver was removed from larvae using flame-sterilized forceps and DNA was extracted using GenCatch™ Plasmid DNA Mini-Prep Kit following the manufacturer’s protocol and stored at -80°C. Extracted DNA was quantified using an Epoch Microplate Spectrophotometer. Viral loads were assessed by real-time qPCR on a StepOnePlus™ Real-Time PCR System using the primers, probe, and thermal cycling protocol designed by Stilwell et al. (2018). For each run all samples were run in duplicate with positive and negative controls along with ten-fold serial dilutions of our FV3 isolate from 10 to 106 PFU mL-1 as standards for sample interpolation. A subset of uninfected controls (N = 6) was analyzed and all tested negative for FV3.
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All analyses were conducted in R Studio v. 2022.02.3 (https://www. rstudio.com/) and significance values were set as α = 0.05. The effect of ranavirus on CTmax was determined using a linear mixed model. Larval CTmax was the response variable with infection treatment, dpi, and their interaction as predictive variables, with larval stage included as a covariate and water bath ID as a random effect. Furthermore, we used Pearson’s correlation analyses to determine the relationships between CTmax and 1) Gosner stage and 2) ranavirus load (PFU/ml). Ranavirus DNA quantities were log10-transformed to achieve normality. Summary values are given as sample size; mean ± 1 standard error.
Southern Illinois University Carbondale