*2.2. Sample Collection*

Fish were sampled after applying the combined stressor of capture and a brief period of confinement, given that stress-induced cortisol levels o ffer a considerable understanding of the overall stress response [44]. In this context, growing evidence suggests that circulating cortisol increases can be detected from as short as 1–2.5 min following exposure to stressors [1,21]. Accordingly, fish were caught using the portable electrofishing unit, and confined in buckets of 20 L for approximately 15 min. Afterwards, specimens were anesthetized with MS-222 (100 mg/L) and immediately after, blood and

skin mucus were sampled. A heparinized insulin syringe was used to collect approximately 0.2 mL of blood by caudal vein puncture. A drop of blood was smeared for hematological analyses, and the remaining fluid kept on ice until transported back to the laboratory. Samples were then centrifuged (1500× *g*, 5 min) and plasma was collected and stored at −20 ◦C. Skin mucus was collected following the method described by Schultz and colleagues [45] with some modifications. Briefly, a polyurethane sponge was used to absorb the skin mucus by applying light pressure to the left and right flank as this method has been shown to be less stressful than using a spatula [45]. The sponge was then introduced into a cylinder of a syringe and compressed with the barrel to collect, into a centrifuge tube, the skin mucus. Afterwards, samples were centrifuged (2000× *g*, 10 min) and the supernatant was stored at −20 ◦C until analysis. Morphological variables, including length (mean ± SD: P1 = 208.5 ± 34.2 mm; P2 = 211.6 ± 22.8 mm; reference = 159.9 ± 35.5 mm) and weight (mean ± SD: P1 = 126.7 ± 61.1 g; P2 = 117.0 ± 50.0 g; reference = 58.7 ± 42.2 g) were measured. Fulton's body condition factor (*K*), calculated according to the formula *K* = 100,000 body weight (g) total length (mm)−<sup>3</sup> [46], was assessed, since it can reflect the energetic state of individuals [47]. Fish were released into the corresponding capture site once the samples had been collected.

## *2.3. Hematological Analysis*

Immediately after being collected, a drop of blood was placed on glass microscope slides, drawn across the surface and, once air-dried, slides were fixed in absolute methanol for 10 min. This procedure was run in duplicate for each specimen. Upon arrival in the laboratory, one of each duplicated slides was stained with Diff-Quick® to assess the frequency of abnormal RBCs and for the WBC count. RBCs (1000) of each individual slide were scored to calculate the frequency of ENA, SE, and IE. The ENAs analyzed were defined as lobed, kidney-shaped, fragmented, and vacuolated nuclei following the directions of Pacheco and Santos [48]. The relative count of all types of WBCs (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) was performed for 100 WBCs following the directions of Tavares-Dias [49]. The neutrophil and lymphocyte count was used to calculate the relative proportion of neutrophils to lymphocytes (hereafter, N:L ratio). The second duplicated slide of each individual was used to assess the number of micronucleus after performing an acridine orange staining. RBCs (3000) of each slide were scored to calculate the frequency of MN.

#### *2.4. Cortisol Extraction and Biochemical Validation*

To analyze cortisol levels from blood and skin mucus, a commercial enzyme immunoassay (EIA) kit (Cortisol ELISA KIT; Neogen® Corporation, Ayr, UK) was used.

Biochemical validation of the EIA was carried out following the methods described by Carbajal and collaborators [50]. Samples of plasma and skin mucus extracts from several individuals were first pooled and used in each validation test. Precision was evaluated with the intra-assay coefficient of variation (CV), calculated from all duplicated samples analyzed. All samples from each matrix evaluated were analyzed in single assays; therefore, the inter-assay CV was not assessed. The dilution test was applied to assess the specificity of the EIA kit by comparing observed and theoretical values from pools diluted with EIA buffer. To test the assay's accuracy, the spike-and-recovery test was used, where known volumes of pools were mixed with different volumes and concentrations of pure standard cortisol solution. Finally, we evaluated the sensitivity of the test, given by the smallest amount of cortisol concentration detected.
