Benzocaine

Salmon lice (Lepeophtheirus salmonis) were reared as earlier described [10 (link)]. After development to the adult stage, 15 lice were removed with forceps from their anaesthetised (80 μg/ml benzocaine) salmon hosts (Salmo salar) and 3 lice were stored in RNA later (Ambion). The remaining 12 lice were starved in incubators with flowing seawater for 14 days. After starvation, 3 lice were sampled and stored as described above, and the remaining 9 lice were put in a tank with uninfected salmon where they could settle on their salmon hosts and resume feeding. After 15 days on their new hosts 3 lice were sampled and stored as described above. The experimental procedures were carried out in accordance with national regulations for use of animals in scientific research.
The transcript levels of LsTryp1 [10 (link)] and the reference gene eEF1α [11 (link)] in 1 selected unstarved, starved and refed lice were determined by quantitative real time PCR carried out with 3 parallels at 5 sequential 2-fold dilutions as previously described [10 (link)]. The RNA purification protocol is previously described [10 (link)] and cDNA syntheses were performed using MultiScribe™ according to the manufacturers recommendations (Applied Biosystems). The Q-PCR results were analysed by the 2^-ΔΔCT method as earlier described [10 (link)] and a method adjusting for PCR efficiency differences described by Peirson et al. [2 (link)]. The latter analysis was performed partially in the DART-PCR Excel spreadsheet [2 (link)]. When using the 2^-ΔΔCT method, at least 2 parallels were required at each dilution. Parallels were removed when the CT value differed more than 0.3 (CT<32) or 0.4 (CT = 32) from the most similar parallel at the same dilution. At least 4 dilutions were required for each stage. The resulting data were calibrated to unstarved lice and analysed as described by Kvamme et al.[10 (link)]. When using the "DART-method", dilutions were removed when PCR efficiency differed significantly (one way ANOVA, α = 0.05) from the other dilutions. The signal corresponding to the initial template concentration (R₀) was derived using the average PCR efficiency for LsTryp1 and eEF1α when the PCR efficiencies were not significantly different (one way ANOVA, α = 0.05). When the PCR efficiency differed significantly, R₀ was calculated using individual gene specific mean efficiencies. The mean R₀ for each dilution of LsTryp1 was normalised to corresponding eEF1α values. The normalised R₀ values were calibrated to the values for unstarved lice. 95% confidence intervals (CI) were derived from normalised R₀ values.

Female silver eels were obtained from a freshwater lake, Vandet, Jutland, Denmark. Male eels were obtained from a Danish commercial eel farm (Stensgård Eel Farm A/S). Experimental maturations were conducted at a DTU Aqua research facility at Lyksvad Fishfarm, Vamdrup, Denmark, where eels were housed in 300 L tanks equipped with a recirculation system [34 ]. Eels were maintained under low intensity light (~20 lux), 12 h day/12 h night photoperiod, salinity of ~36 ppt, and temperature of 20°C. Acclimatization took place over 10 days. As eels naturally undergo a fasting period from the onset of the pre-pubertal silvering stage, they were not fed during treatment. Prior to experimentation, eels were anaesthetized (ethyl p-aminobenzoate, 20 mg L^-1; Sigma-Aldrich, Missouri, USA) and tagged with a passive integrated transponder. Females used for experiments (n = 4) had a mean (± SEM) standard length and body weight of 65 ± 4 cm and 486 ± 90 g, respectively. To induce vitellogenesis females received weekly injections of salmon pituitary extract (Argent Chemical Laboratories, Washington, USA) at 18.75 mg kg^-1 body weight [11 (link), 34 ]. To stimulate follicular maturation and induce ovulation, females received 17α,20ß-dihydroxy-4-pregnen-3-one (Sigma-Aldrich, Missouri, USA) at 2 mg kg^-1 body weight [35 ] and were strip-spawned within the subsequent 12–14 h. Male eels (n = 11) had a mean (± SEM) standard length and body weight of 40 ± 3 cm and 135 ± 25 g, respectively. Males received weekly injections of human chorionic gonadotropin (Sigma-Aldrich, Missouri, USA) at 150 IU per fish [34 ]. Prior to fertilization, an additional injection was given and milt was collected by strip-spawning ~12 h after administration of hormone. Milt samples were pipetted into a P1 immobilizing medium [36 (link)] and only males with sperm motility of category IV (75–90%) were used for fertilization within 4 h of collection [37 ]. Only floating viable eggs/embryos were further used for experimentation.

We tested the effect of four anaesthetic agents commonly used in fish: (i) 2-phenoxyethanol (2-PE; 1-hydroxy-2-phenoxyethane; Sigma-Aldrich, Steinheim, Germany), (ii) ethyl-3-aminobenzoate methanesulfonate (also known as tricaine, TMS or MS-222; Sigma-Aldrich, Steinheim, Germany), (iii) benzocaine (Sigma-Aldrich, Steinheim, Germany; solved 1:10 in 95% ethanol), and (iv) Aqui-S (Scanvacc, Hvam, Norway). The use of one of these anaesthetics in a given experiment was selected at random to ensure that any differences could not be caused by unintended changes in the experimental setup or by undetected changes in the animals’ state. Each of the experimental fish was solely exposed to one of the anaesthetic agents to exclude potential interactions between the anaesthetics. Anaesthetic concentration levels, particularly surgical concentrations, were chosen based on appropriate references^{4 ,21 (link),23 ,26 –29 (link)}. For the appropriate use of Aqui-S, an anaesthetic widely used in aquaculture facilities, we additionally used information provided by Aqui-S New Zealand Ltd (http://www.aqui-s.com/aqui-s-products/aqui-s) (2018). Concentration levels ranged from 0.2 to 1.0 ml L⁻¹ for 2-PE, 20 to 100 mg L⁻¹ for MS-222, 20 to 150 mg L⁻¹ for benzocaine, and 10 to 20 mg L⁻¹ for Aqui-S. 20 mg L⁻¹ of the anaesthetics MS-222 and benzocaine cause slight anaesthetisation in fish (stage II anaesthesia^{1 ,20} ), whereas all other applied anaesthetic concentrations cause at least surgical anaesthetisation (stage III anaesthesia)^{21 (link)}. Before starting any surgical intervention, fish were surgically anaesthetised (stage III anaesthesia^{1 ,20} ) by application of either 0.4 ml L⁻¹ 2-PE, 60 mg L⁻¹ MS-222 or benzocaine or 20 mg L⁻¹ Aqui-S for 15 min. We generally confirmed the sufficiency of the anaesthetisation after total loss of equilibrium by carefully exerting pressure to the fish’s caudal peduncle. In responsive fish, this kind of touch reliably triggers an escape response and subsequent swimming behaviour. When this stimulation (and subsequent handling) yielded no response, then the fish was positioned in the electrophysiological recording chamber and artificial respiration was established with aerated water flowing via a tube through the mouth and out over the gills at a flow rate of 80 ml min⁻¹. Respiration water thereby was delivered to the fish from a reservoir (respiration water tank) using a suitably adjusted pump (EHEIM universal 300; EHEIM GmbH & Co. KG, Deizisau, Germany; regular power: 300 L h⁻¹, adjusted to 4.8 L h⁻¹). To maintain anaesthesia, the respiration water always contained the same anaesthetic as used for establishing anaesthesia. We started experiments randomly either with the lowest concentration used in the respective experiment or with the highest one. To examine anaesthetic concentration effects, we then changed the concentration level within a particular animal, while simultaneously recording intracellularly from the Mauthner neuron. To increase the concentration level during the experiment, we added additional anaesthetic to the respiration water. To quickly establish a uniform mixture of respiration water and anaesthetic, we used a circulation pump (EHEIM universal 600; EHEIM GmbH & Co. KG, Deizisau, Germany; power: 600 L h⁻¹) in the respiration water tank. To reduce the anaesthetic concentration, we added additional water of the same quality and temperature to the respiration water tank. After changing the anaesthetic concentration level, we always gave an acclimatisation period of 15 min before the next measurements were taken. This interval was chosen to be significantly beyond the estimated time (<6 min) needed by the used anaesthetics to impact on the animal’s physiology by simple add-on to the water surrounding the fish^{23 ,27 (link)}.

Ethyl-4-aminobenzoate (500 mg, 3.0
mmol) and propionic acid (5 μL) were dissolved in water (50
mL). The solution was cooled to 0 °C, and ethylene oxide (5.8
mL of a 1.13 M solution in methanol, 6.60 mmol) was added dropwise.
The mixture was stirred at room temperature overnight. The compound
was extracted with dichloromethane (3 × 30 mL) and washed with
a saturated aqueous solution of sodium bicarbonate (2 × 20 mL).
The solvent was removed under reduced pressure. The crude product
was recrystallized in methanol. The compound was dried under vacuum.
Yield: 516 mg (2.0 mmol, 68%). ¹H NMR (500 MHz, CD₂Cl₂): δ 7.78 (d, J = 8.1
Hz, 2H), 6.91 (d, J = 8.1 Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 4.09 (t, J = 5.3 Hz, 4H),
3.67 (t, J = 5.3 Hz, 4H), 1.37 (t, J = 7.2 Hz, 3H). ¹³C{¹H} NMR (125 MHz, CD₂Cl₂): δ 165.7, 151.6, 129.1, 117.3, 111.6,
62.4, 61.2, 59.7, 15.2. HR-MS (m/z): [M + H]⁺ calcd for C₁₃H₂₀NO₄, 254.1392; found, 254.1391.

Juvenile rainbow trout (15.47 g ± 0.88) were obtained from Pisciculture Rio Blanco (Pontificia Universidad Católica de Valparaiso, Valparaíso, Chile). Fish were held under a temperature of 14 ± 1 °C and photoperiod conditions of L/D 12:12. Juvenile fish were fed with Skretting pellets. Fish were sedated with benzocaine (25 mg/L) and intraperitoneally injected with metyrapone (Sigma-Aldrich, St. Louis, MO, USA) (1 mg/kg of fish) for one hour and then divided into different groups. Fish in the first and second groups were treated with vehicle solution (DMSO, PBS 1x) and 11-deoxycorticosterone acetate (DOC, USBiological, Salem, MA, USA), respectively, at physiological concentrations (1 mg/kg). Fish in the third and fourth groups were treated with mifepristone (RU486, Sigma-Aldrich) (1 mg/kg) and mifepristone (1 mg/kg) plus DOC (1 mg/kg), respectively. Finally, fish in the fifth and sixth groups were treated with eplerenone (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1 mg/kg) and eplerenone (1 mg/kg) plus DOC (1 mg/kg), respectively. Three hours after treatment, all rainbow trout (n = 24, four fish per group) were euthanized with benzocaine (Veterquimica, RM, San Bernardo, Chile) (300 mg/L). Heparinized obtained blood was centrifugated at 5000× g for 10 min and the plasma was stored at −80 °C. Myotomal skeletal muscle was isolated from the epaxial area, frozen in liquid nitrogen for 6 h, and then stored at −80 °C.

Each fish was stimulated to increase the number of metaphases with an injection of yeast suspension [29 (link)] in the caudal peduncle 48 h before injecting intra-abdominally a dose of 0.01 mL/g colchicine (0.0125%). After colchicine treatment, fish were maintained at room temperature for 24–48 h in a well-aerated aquarium and finally euthanized with an overdose of benzocaine [30 ].
Cell suspensions containing mitotic chromosomes were obtained from the animals’ kidneys [31 ]. Chromosomes were stained with 5% Giemsa solution (phosphate buffer, pH 6.8) to define the diploid numbers (2n) and karyotype formula. Heterochromatic regions were identified by the C-banding procedure [32 (link)]. Nucleolus organizer regions (NORs) were stained following silver nitrate impregnation [33 (link)].
The fluorescence in situ hybridization experiments were performed according to Pinkel et al. [34 (link)], with some adaptations described in Soares et al. [35 (link)] and Sassi et al. [36 (link)]. In summary, metaphase chromosomes were treated with RNAse A (40 μg/mL) for 1.5 h at 37 °C and denatured in 70% formamide/2× SSC at 72 °C for 3 min. The hybridization mixture (2.5 ng/μL probes, 50% deionized formamide, 10% dextran sulfate) was applied to the slides and hybridization was performed for 14 h at 37 °C in a dark moist chamber. The probes of major and minor ribosomal genes (18S rDNA, 5S rDNA), and telomeric sequence (TTAGGG)n were obtained by polymerase chain reaction (PCR) using primers described by Utsunomia et al. [37 (link)], Pendas et al. [38 (link)], and Ijdo et al. [39 (link)], respectively. These probes were directly labeled through Nick-Translation, using Atto488-dUTP (18S rDNA) or Atto550-dUTP (5S rDNA and telomeric sequence), according to the manufacturer’s instructions (Jena Biosciences, Jena, Germany). Chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in antifading solution (Vector Laboratories, Burlington, ON, Canada) after a post-hybridization wash in 1× SSC at 65 °C and 4× SSC/Tween at room temperature for 5 min each.
For each individual, at least 60 metaphases were recorded to determine the diploid modal number and confirm the FISH results, from which the best 20 to 30 mitotic figures were chosen to acquire photographs.