Liver sampling

Twenty juvenile tambaquis were collected ex-situ from two fish farms located in the northern and southeastern regions of Brazil (Figure 1). Sampling was carried out during the dry season when regional climate variables were similar between both sites. Thena (n= 10; ~ 26 g and 1 population from Balbi0 cm) was collected in June 2016, at the beginning of the Amazonian “summer” period (Fisch et al., 1998), with temperatures varying between 23 to 31ºC (Climatempo, 2019). The population from Brumado (n= 10; ~ 60 g and 13 cm) was collected during the summer of February 2016, when temperature varied from 18.8 to 28ºC (CPTEC/INPE, 2019). At the time, the water temperature of the rearing tanks was 29.5ºC in Balbina and 21ºC in Brumado; the level of dissolved oxygen ranged from 5 to 7 mg.L^-1.

For tissue sampling from each population, fish (42 g ± 4.7 and 11 cm ± 0.4) were anesthetized and euthanized by cervical sectioning according to Brazilian Guidelines from the National Board of Control and Care for Ethics in the use of Experimental Animals (CONCEA, 2013). Twenty liver samples were immediately stored in RNAlater® Stabilization Solution (Thermo Fisher Scientific, Massachusetts, USA) to ensure the preservation of the ribonucleic acid (RNA) during transport to the Laboratory of Ecophysiology and Molecular Evolution (LEEM/COBio/INPA), Manaus, Amazonas state, Brazil. In the laboratory, samples were removed from RNAlater®, washed in RNase-free water (Qiagen, Hilden, DE), dapped dry on an absorbent paper tissue (Whatman, GE Healthcare Life Sciences, Maidstone, UK), and then stored at -80 ºC until extraction of the RNA. Herein, the liver was analyzed tissue due to its essential metabolically responses under environmental stress (Lemgruber et al., 2013; Logan and Buckley, 2015).