In vivo exposures to actinospores plus compounds

After quantification using the enumeration technique described earlier, we added 50 mL aliquots of ~30 000 M. cerebralis actinospores to 1 L containers. This was followed by adding 50 mL of each salt solution at twice the concentration from the tolerance test, to make 100 mL of exposure solutions at the desired salt concentration (Table 1). The temperature of all exposure solutions was 16–20°C. To better understand the effect of divalent cations on nematocyst discharge in M. cerebralis, we included MgCl₂ as a treatment in in vivo. We also included KMnO₄, an oxidizing agent used to treat parasite infections in aquaculture (Straus and Griffin, 2002). Positive controls (fish with actinospores and no salt) and negative controls (fish with neither actinospores nor salt) were included.

The following steps were used to expose fish to M. cerebralis. First, exposure solutions were equilibrated for ~15 min prior to the addition of fish. In parallel, treatment groups of 3 fish were acclimated in 1 L aerated containers with 100 mL of each salt solution for 5 min in the absence of parasites. Groups of acclimated fish were each netted and transferred to the 1 L container with the corresponding salt and actinospore solution. Fish were exposed to spores for 15 min, netted, then dipped three times in a spore-free solution of the appropriate salt to rinse off poorly adhered actinospores. Each group of fish was then returned to the container of parasite-free solution used for the original acclimation, held for 5 min, then the fish were killed by an overdose of buffered MS-222 in the salt. Gills were removed and frozen for downstream processing. Of the known sites of attachment for M. cerebralis, gills were chosen as they accumulate more spores than skin (Eszterbauer et al., 2019) and we suspected their rate of actinospore encounter might be less variable between fish than fins. Instruments were decontaminated with hydrogen peroxide between samples. The experiment was conducted twice, with different batches of spores from the same stock solutions and a cohort of unexposed fish.

DNA was extracted from each gill sample using a Qiagen DNeasy Blood and Tissue kit following the manufacturer's recommendations, with DNA eluted from the column with two washes of 100 μL buffer AE. Total DNA concentration was measured with a Nanodrop spectrometer (Thermo Scientific) and normalized by dilution with molecular grade water to 100–250 ng/μL, to achieve an optimal range of detection. M. cerebralis DNA was quantified using an Applied Biosystems StepOnePlus Real-Time PCR System and the amplification profile and M. cerebralis-specific TaqMan probes and primers described by Kelley et al., 2004. Each 10 μL reaction contained 1.79 μL molecular grade water, 0.15 mm bovine serum albumin, 80 nm TaqMan probe, 400 nm each primer, 1 × TaqMan Universal PCR MasterMix, and 2 μL diluted gDNA. In a second qPCR using the same machine, we measured the concentration of a single-copy rainbow trout gene for endogenous normalization. Each 10 μL reaction contained 1 μL molecular grade water, 1 × SYBR Green Power Master Mix, 4 μm of each ‘gDNA Salmonid EF1-α’ primers (Bland et al., 2012) and 2 μL gDNA further diluted 1:10. Amplification proceeded with a 10 min incubation at 95°C and then 40 cycles of 95°C for 15 s and 60°C for 1 min. For both reactions, samples were run in triplicate wells, and included a positive control (DNA extracted from M. cerebralis actinospores and rainbow trout, respectively) and a negative control (molecular grade water).