DOI: 10.5061/dryad.4f4qrfjj7
title: Rapid hyperthyroidism-induced adaptation of Salmonid fish in response to environmental pollution

description: The streams draining volcanic landscapes are often characterized by a
 complex of factors that negatively affect hydrobionts and lead to a
 decline of their populations. However, in a number of cases, hydrobionts
 ensure the resilience of populations througth a range of rapid adaptive
 changes. Here, we present both field and experimental data shedding light
 on the physiological basis of adaptation in populations of Dolly Varden
 charr (Salvelinus malma) differing in the duration of isolation in
 volcanic streams contaminated with heavy metals. The study reveals that
 isolated populations have a physiological phenotype that distinguishes
 them from the populations inhabiting clean waters. They are characterized
 by a hyperthyroid status accompanied by an increased metabolic rate,
 elevated activity of antioxidant enzymes, decreased ionic conductivity of
 tissues and reduced stored energetic reserves. The experimental data
 elucidate that hyperthyroidism is an adaptive characteristic enhancing the
 resistance to heavy metal contamination and shaping the evolution of these
 populations. The similarity of physiological, developmental and
 morphological changes in isolated populations suggests a common source and
 mechanisms underpinning their “evolutionary rescue”. Thus, populations of
 S. malma trapped in volcanic streams represent a genuine case of a rapid
 neuroendocrine-driven adaptation to changing environmental stimuli.
description: Immediately after sampling,
 blood was sampled from the caudal vessel of fish using vacutainers with
 K2EDTA (Galen). Then, the blood was centrifuged for 3 min under 1900 g
 (velocity-6µ, Dynamica), and a supernatant (plasma) was collected and
 frozen for further hormonal (cortisol and total T<sub>3</sub>)
 content analysis. The left lateral body muscle, peeled from skin and
 blood, and a caudal fragment of the liver (both 0.10 ± 0.01 g) were
 sampled for the analysis of stored energetic resource by assessing of the
 triglycerides and glycogen concentrations, respectively (Schlenk et al.,
 2008). The remaining part of the body was gutted (removal of coelomic
 organs) and frozen at -50⁰C for further analysis of the activity of
 antioxidant enzymes and ATP-ase ion pumps.
 The set of biochemical
 parameters was measured spectrophotometrically using the photometer StatFax 303 Plus (Awareness Technology). The
 fish tissues were homogenized using TissueLyser (Qiagen), centrifuged for
 5 min at 1900 g (velocity-6µ, Dynamica), and the supernatant was
 collected.
 <strong><em>Enzyme
 activity</em></strong><strong><em>.</em></strong> The fish were homogenized in 2 mMol EDTA buffer (pH = 7.2) with 50 mMol Imidazole (cas 288-32-4) and 2 mMol Triton X-100 (cas 9002-93-1) in a mass ratio 1:3. After 15-min incubation at 4°С and 3-min centrifugation, the supernatant was sampled. The precipitate was vortexed in EDTA buffer (1:3), incubated in cold for 5 min and centrifuged again. The pooled supernatant was split in half for two analyses. The activity of antioxidant enzymes (mainly catalase) was evaluated following Koroluk et al. (1988). The supernatant was added in a ratio 1:20 to 0.01% peroxide in PBS with 1 mMol EDTA and incubated at 22°С for 20 min. Then, the incubation medium was diluted 1.5 times with the stop-reagent (2% paramolybdate (cas 12027-67-7) in PBS with 2% PFA, 4°С), centrifuged for 2 min and read at 405 nm. The activity of ATP-ase ion pumps was evaluated following Zaugg (1982) by the difference in the inorganic phosphate concentration in the incubation medium versus individual control. The working supernatant was added in a ratio 1:10 to Milli-Q water containing 30 мMol MgCl<sub>2</sub>, 30 mMol Ca(СО<sub>3</sub>)<sub>2</sub>, 1300 мМol NaCl, 200 мМol KCl and 30 мMol ATP. The control supernatant was added in the same ratio to Milli-Q water containing 50 мMol Rabeprazole (cas 117976-89-3) and 20 mMol Cinnarizin (cas 298-57-7). After 20-min incubation at 22°С, 0.2 volume of the stop-reagent (10% sodium acetate in PBS with 2% PFA, 4°С) was added to both incubation mediums. The resulting solutions were centrifuged for 2 min and the phosphate content was assessed using MAK308 kit (Sigma Aldrich) based on a proprietary formulation of the malachite green dye (read at 630 nm). Both obtained activities were recalculated to the sample weight. <strong><em>Triglycerides.</em></strong> Following Folch et al. (1957) procedure, lipids were extracted from the shredded muscle samples and purified with 1% KCl solution employed as the aqueous phase. The enzymatic hydrolysis reactions (Spinreact kits) provided a triglycerides (TAGs) value (Trinder, 1969). The indicator substance kinonimin was measured at 505 nm. <strong><em>Glycogen.</em></strong> The liver samples were placed in 2 ml 30% NaOH at 100°C for 30 min. Then, 0.5 ml of 60% H<sub>2</sub>SO<sub>4</sub> was added to the sample, and the volume was brought up to 1 ml with Milli-Q water. The sample was mixed with 7.5 ml of 0.2% Anthrone reagent (cas 90-44-8, Sigma Aldrich) and incubated at 100°C for 20 min (Templeton, 1961). The optical density was measured at 630 nm against glycogen standards. <strong><em>TBARS.</em></strong> Malondialdehyde was obtained from 0.2 ml supernatant by adding 1 ml of thiobarbituric acid in PBS, subsequent incubation at 100°С for 45 min and 10-min centrifuging with 4 ml of butanol. The supernatant concentration was determined at 570 and 535 nm (Gavrilov et al., 1987). <em><strong>Hormones.</strong></em> We measured the total T<sub>3</sub> and cortisol content in plasma of natural fish. To measure the T<sub>3</sub> content in the whole body of experimental fish, we used barbiturate-extraction protocol (Holzer et al., 2017). The hormonal content was evaluated by enzyme-linked immunosorbent assay (ELISA) with commercially available Monobind (T<sub>3</sub>) and Xema (cortisol) kits in accordance with the manufacturer’s protocols.
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