Benzocaine

Laying beds were placed into harem tanks containing one male per two females the night before embryo collection. Embryos were collected in Petri dishes following light-induced spawning and sorted to remove non-viable offspring, then routinely transferred into 0.003% w/v 6-n-propyl thio-uracil (PTU) (Sigma-Aldrich, Australia) in system water and incubated at 28°C. Time-staged embryos were humanely sacrificed by adding 10% Benzocaine (Sigma-Aldrich) drop-wise to Petri dishes containing embryos, after which the embryos were transferred to Trizol (Life Technologies, Australia) for RNA extraction or fixed in 4% paraformaldehyde (PFA) for in situ hybridization. Alternatively, specific organs and tissues were dissected from humanely sacrificed adult zebrafish and transferred to Trizol.

At 90 dpf, a minimum of 50 fish per batch were randomly sampled and euthanized with an overdose (200 mg/L) of benzocaine (Sigma-Aldrich). The sex was determined using the aceto-carmine squash method for sexing juvenile fish [27 ]. A piece of gonad was removed, stained with a few drops of aceto-carmine, squashed with a cover slide and observed with an optical microscope (40× magnification). Phenotypic sex was assessed based on the presence of oocytes in the females and on the lobular morphology of the testis for the males. Analysis of the proportion of males was performed using the generalized linear model [26 ]. Post hoc comparisons between treatments for each progeny were assessed using Tukey’s test within the least-squares means R package [25 ].

Female Xenopus laevis were purchased from Hamamatsu Seibutsukyozai (Shizuoka, Japan) and maintained in dechlorinated water at 18°C. Oocytes were surgically obtained following anesthesia of a Xenopus frog using 0.03% benzocaine (Sigma-Aldrich, Tokyo, Japan), and enzymatically defolliculated by incubating them for 1–1.5 h in a Ca²⁺-free SOS containing 1 mg/ml collagenase (Wako Pure Chemical Industries, Osaka, Japan). Vssc cRNA and tipE cRNA (0.9 ng each) were co-injected into healthy stage V–VI oocytes, which were incubated at 18°C in SOS medium supplemented with 50 µg/ml gentamycin, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 mM Na pyruvate, and 4% horse serum, for 2–4 days after injection. The medium was replaced daily and unhealthy oocytes were discarded. It is generally recognized that the knockdown resistance is a recessive trait [19] –[21] . Since we injected mRNA of a single Vssc haplotype into Xenopus oocytes, theoretically we reproduced the environment of the homozygous Vssc form in the cell.

Rainbow trout were killed by benzocaine (Sigma-Aldrich) overdose and the spleen collected. Single cell suspensions were obtained as previously reported^{18 (link),24 (link)} using 100 µm nylon cell strainers (BD Biosciences) and Leibovitz medium (L-15, Invitrogen) supplemented with 100 I.U./ml penicillin and 100 µg/ml streptomycin (P/S, Life Technologies), 10 units/ml heparin (Sigma) and 5% foetal calf serum (FCS, Life Technologies). Cell suspensions were placed onto 30/51% discontinuous Percoll (GE Healthcare) density gradients and centrifuged at 500 × g for 30 min at 4 ºC. The interface cells were washed twice in L-15 with 2% FCS and cells were resuspended in L-15 with 5% FCS. The viable cell concentration was determined by Trypan blue (Sigma-Aldrich) exclusion, adjusting the concentration to 2 × 10⁶ cells/ml.

Rainbow trout were killed with benzocaine (Sigma-Aldrich) and blood was extracted with a heparinized needle from the caudal vein. Blood was diluted 10 times with Leibovitz's medium (L-15, Gibco) containing 100 I.U./ml penicillin and 100 μg/ml streptomycin (P/S, Life Technologies), 10 I.U./ml heparin and 5% fetal calf serum (FCS, Thermo Fisher Scientific) and then placed onto 51% Percoll (GE Healthcare) density gradients. Cell suspensions were centrifuged at 400 × g for 30 min at 4°C, the interface cells were collected and washed with L-15 supplemented with antibiotics and 5% FCS. The viable cell concentration was determined by Trypan blue (Sigma-Aldrich) exclusion and cells were resuspended in L-15 with 5% FCS at a concentration of 2 × 10⁶ cells/ml.

One-year old Atlantic cod, Gadus morhua L., reared in land-based tanks supplied with filtered sea water were obtained from Ardtoe Marine Facility (UK) during 2014-2015. They were kept at environmental temperature and fed on commercial dry pellets once a day. All procedures were in accordance with UK Home Office welfare guidelines. The fish were sacrificed with UK Home Office approved methods, using an overdose of the anesthetic benzocaine (Sigma-Aldrich) followed by brain concussion. The head kidney and spleen of each animal were dissected out and kept at 4°C in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich) and 0.2% Primocin™ (InvivoGen) until cell culture.

In preliminary experiments, an appropriate tracer incorporation period for the flooding dose method of measuring fractional rates of protein synthesis (Lamarre et al. 2015 (link)) was determined for rainbow trout held at 13 °C. Fish (N = 15) were lightly anaesthetized in a solution of benzocaine (50 mg L⁻¹ ethyl-p-aminobenzoate; Sigma-Aldrich, Oakville, ON, Canada), and mass and fork length were measured. Fish were placed in 4 L individual Plexiglass chambers supplied with flowing, aerated 13 °C water. Following an overnight recovery period, unanesthetized fish were given an intra-peritoneal injection of 150 mM phenylalanine containing 50% ring-D₅l-phenylalanine (D₅-PHE, 98%; Cambridge Isotope Laboratories, Andover, MA, USA) in distilled water at a dosage of 1 mL 100 g⁻¹. After 0, 60, 120, 240 or 360 min, fish (N = 3 at each time) were rapidly euthanized via immersion in a terminal anaesthetic solution (500 mg L⁻¹ ethyl-p-aminobenzoate). The peritoneal cavity was immediately exposed and rinsed with distilled water to wash away unabsorbed tracer. Liver and white muscle samples were collected, flash frozen in liquid nitrogen, and stored at – 80 °C until analysis.