Stressor‐ and Tissue‐Specific Regulation of the Corticotropin‐Releasing Factor System Across Epithelial Tissues in Rainbow Trout

Brett M Culbert; Alexis E Pulford‐Thorpe; Carol Best; Nicholas J Bernier

doi:10.1096/fj.202603109R

. 2026 Sep 5;40(17):e72270. doi: 10.1096/fj.202603109R

Stressor‐ and Tissue‐Specific Regulation of the Corticotropin‐Releasing Factor System Across Epithelial Tissues in Rainbow Trout

Brett M Culbert ^1,^✉, Alexis E Pulford‐Thorpe ¹, Carol Best ¹, Nicholas J Bernier ^1,^✉

PMCID: PMC13545658 PMID: 42698382

ABSTRACT

The corticotropin‐releasing factor (CRF) system is a major neural regulator of stress responses in vertebrates. However, stress‐related roles for the CRF system in other tissues—and whether these roles vary between stressor types—remain unclear. To address this gap, we first characterized the CRF system in the gills and intestine of rainbow trout ( Oncorhynchus mykiss ) and then evaluated how it is transcriptionally regulated following either an immune (vaccination) or osmotic (seawater transfer) stressor. Additionally, since the CRF system is involved in food intake regulation, we also evaluated whether feeding state affects the intestinal CRF system. Vaccination against Vibrio anguillarum caused increases in intestinal CRF binding protein transcripts paired with reductions in ligand (crfa2) and receptor (crfr1b) transcripts but did not affect transcript levels of CRF system components in the gills. In contrast, seawater transfer caused the abundance of most CRF system transcripts to increase in the middle (but not posterior) portion of the intestine, while transcript levels of CRF binding proteins and receptors in the gills declined. Finally, levels of CRF system transcripts in the intestine varied with feeding state in a region‐specific manner. In the middle intestine, transcript levels of most components declined with fasting and increased when feeding was resumed, whereas the opposite pattern occurred in the posterior intestine. Overall, our results implicate the peripheral CRF system as a stressor‐ and epithelial tissue‐specific modulator of immune and osmoregulatory functions in teleosts.

Keywords: fasting, gills, immune response, intestine, osmotic stress

The gill and intestinal corticotropin‐releasing factor (CRF) systems in rainbow trout are differentially regulated by immune, osmotic, and nutritional stressors. Seawater transfer suppressed gill CRF system transcription and induced time‐dependent changes in the middle intestine. An immune challenge (vaccination) altered only intestinal CRF expression. Fasting and refeeding triggered region‐specific intestinal responses. These findings reveal extensive tissue‐ and region‐specific regulation of the CRF system, highlighting its diverse roles in coordinating stress responses beyond the brain. Images created using Google Gemini.

graphic file with name FSB2-40-e72270-g008.webp

1. Introduction

The ability to appropriately respond to environmental stressors is critical for an animal's survival and fitness. To accomplish this, a suite of physiological responses occurs following a stressor [1, 2], including elevated levels of two major classes of hormones: catecholamines (e.g., adrenaline and noradrenaline) and corticosteroids (e.g., cortisol and corticosterone). While catecholamines are critical for rapid physiological changes associated with “fight‐or‐flight” responses (seconds to minutes; [3, 4]), contributions of corticosteroids are generally slower and more prolonged (hours to days), helping animals adjust their physiology—including metabolic, immunologic, osmotic, and neural responses [5, 6]—to better cope with, overcome, and/or recover from a stressor [7]. Given the relative ease with which corticosteroid levels can be measured and manipulated, a major focus of ecological and physiological studies alike has been to understand the regulation of and roles for these hormones during vertebrate stress responses [8, 9, 10]. However, it is becoming increasingly clear that many hormone systems are necessary to overcome stressors and corticosteroids—while important—are but one of several hormones involved in coordinating stress responses [11, 12, 13]. Furthermore, the relative contribution of different hormone systems during stress responses varies depending on the nature, intensity, and duration of a stressor [14, 15, 16, 17]. Yet, most studies continue to solely focus on the regulation of corticosteroids in response to environmental stressors and the relative contribution(s) of other stress‐related hormone systems remain poorly understood.

The corticotropin‐releasing factor (CRF) system—consisting of five ligands [CRFa, CRFb, urotensin 1 (UTS1), urocortin 2 (UCN2), and UCN3], a binding protein (CRFBP), and two receptors (CRFR1 and CRFR2) in teleost fishes [18, 19]—is the major neuroendocrine regulator of stress responses via its effects on the hypothalamic–pituitary–adrenal/interrenal axis and corticosteroid synthesis [5, 20]. However, the CRF system also contributes to the regulation of many physiological processes outside of the brain. For example, activation of the CRF system stimulates proinflammatory responses in mammals [21, 22, 23], counter to the anti‐inflammatory effects that are generally associated with corticosteroids [24]. Similarly, the CRF system is an important regulator of osmoregulatory functions in the mammalian colon where it reduces ion and water absorption [21, 25]. As in mammals, previous work in fish has suggested that the CRF system is involved in coordinating responses to immune challenges [26, 27, 28, 29] and osmotic disturbances [30, 31, 32, 33, 34, 35, 36]. However, most of these studies have focused on only a small subset of CRF system components, and only Mazon et al. [27] evaluated whether responses of the CRF system vary in a stressor‐ and tissue‐specific manner using common carp ( Cyprinus carpio ). Therefore, we set out to compare whether transcriptional regulation of the CRF system varies between immune and osmotic stressors. We specifically focused on the gills and intestine because both tissues are key osmoregulatory organs [37, 38, 39] and are important for the prevention of pathogen entry from the external environment [40, 41].

To accomplish this, we first characterized which components of the CRF system were most abundant in the gills and intestine of rainbow trout ( Oncorhynchus mykiss ). We then evaluated whether transcript levels of the major CRF system components in the gills and intestine were affected by either an immune (6, 24, 72, and 168 h post‐vaccination) or osmotic (24, 72, and 168 h post‐SW transfer) stressor. We predicted that vaccination would initially stimulate CRF system activity in the gill and intestine (e.g., greater transcript abundance of CRF ligands and receptors, paired with reductions in CRFBP), associated with the initial production of proinflammatory cytokines, followed by a suppression of CRF system activity (e.g., reduced transcript abundance of CRF ligands and receptors, paired with increases in CRFBP) after production of anti‐inflammatory cytokines increases. Following SW transfer, we predicted that gill CRF system activity would increase (reflecting the development of an ion‐secretory phenotype) and that intestinal CRF system activity would decrease (reflecting a heightened need for ion and water absorption). Additionally, because the CRF system is a major regulator of food intake in teleosts [42, 43, 44, 45] and salmonids naturally suppress feeding in response to stress—including osmotic and immune stressors [46, 47, 48]—we also assessed whether feeding status influences transcript abundance of the intestinal CRF system. Specifically, we compared transcript abundance of CRF system components in the intestine of fed fish with fish fasted for 0 (fed), 24, 72, or 168 h, as well as 24, 72, or 168 h after resumption of feeding following a 168 h fast. Here, we predicted that activity of the intestinal CRF system would decline during fasting to maximize nutrient absorption capacity (i.e., reduced secretory capacity).

2. Methods

2.1. General Housing

All trout were acquired from the Ontario Aquaculture Research Centre (Alma, ON, Canada) and were housed in the Hagen Aqualab at the University of Guelph (Guelph, ON, Canada). Fish were initially maintained in 1.8 m diameter fiberglass tanks (~2000 L) supplied with flow‐through well water maintained at 12°C and kept on a 12 h light, 12 h dark photoperiod regime. Fish were fed to satiation three times per week with commercial trout food (3 or 5 PT Sinking, Blue Water Fish Food; Guelph, ON, Canada). A stocking density of ~100 fish per tank was maintained during this period, and fish were kept under these conditions for at least a month prior to experiments. All procedures were carried out in accordance with the Canadian Council for Animal Care guidelines for the use of animals in research and teaching and were approved by the University of Guelph's Animal Care Committee (AUPs #4123 and 4996).

2.2. Evaluating the CRF System of the Gills and Intestine

To determine which components of the CRF system were most abundant in the gills, as well as the middle (Mid. Int.) and posterior (Post. Int.) portions of the intestine, we sampled three sexually immature trout (mass = 156 ± 10 g, fork length = 23.3 ± 0.9 cm) and performed qPCR (see Section 2.5) using gene‐specific primers for each component (Table 1). Values for each transcript were corrected for primer efficiency and are expressed relative to the abundance of crfa2 in the gills for visualization purposes, which was the CRF system component with the lowest levels across both tissues that consistently amplified. We measured individual paralogs of all CRF system components, except for UCN3, which shares 99% identity across paralogs.

TABLE 1.

Gene specific primers used for real‐time polymerase chain reaction (qPCR).

	Primer sequence (5′ to 3′)	Amplicon size (bp)	Efficiency (%)	Accession number
crfa1	F: CAAAATTCGCTCCAGTCCTC	95	100	XM_021613200
crfa1	R: TGCGTGAGCTGAAGTTGTAA	95	100	XM_021613200
crfa2	F: ATTCGCCCCAATCTTCATT	79	102	XM_021589550
crfa2	R: TGAAGTAAAGCCCTGTTGACC	79	102	XM_021589550
crfb1	F: CTGAAGGCATGTACCCAGAG	110	102	NM_001124286
crfb1	R: GACGAACCGGCTGATGTT	110	102	NM_001124286
crfb2	F: GAAAGTCATGCACCCCAAG	117	102	NM_001124627
crfb2	R: AAGCCGCTGATGAACCTATT	117	102	NM_001124627
crfbp1	F: CCAACACGTTGTCTATCTAC	107	100	NM_001124631
crfbp1	R: TTTTATGGCAACCTTCAGGCT	107	100	NM_001124631
crfbp2	F: CTCCCTATCTATCTCTCCAC	122	100	XM_036977080
crfbp2	R: GTTATGGCGATCCTCAGGAT	122	100	XM_036977080
crfr1a	F: TCCCTCGGAGACAGGAGTGT	121	102	XM_036940770
crfr1a	R: CTTTTACGATGTGCGACTGGT	121	102	XM_036940770
crfr1b	F: CGGCAATTTATAGGCTGTGTT	114	99	XM_021624572
crfr1b	R: CCCACATGAACAACCAACAG	114	99	XM_021624572
crfr2a	F: CACTGTACTGTACTAGTGGT	83	100	XM_021613636
crfr2a	R: TCCTGCTTAAATGATCTCTGC	83	100	XM_021613636
crfr2b	F: TAAATTCAGAACAGATGGGC	82	100	XM_021589160
crfr2b	R: CATTGCCAGTAAGACTCTGAC	82	100	XM_021589160
c3	F: GAGCAGCACCTGGCTGACA	150	102	XM_021561577
c3	R: GGAGCAAACTCATTGAAGATGCC	150	102	XM_021561577
ef1α	F: CCATTGACATTTCTCTGTGGAAGT	106	96	NM_001124339
ef1α	R: GAGGTACCAGTGATCATGTTCTTGA	106	96	NM_001124339
ifnγ	F: ACCCTGTTTTCCCCAAGGAC	61	92	NM_001124620
ifnγ	R: ACCACACTCATCAACACCCTC	61	92	NM_001124620
il1b	F: TGAGAACAAGTGCTGGGTCC	148	107	NM_001124347
il1b	R: GGCTACAGGTCTGGCTTCAG	148	107	NM_001124347
il10	F: CCGCCATGAACAACAGAACA	105	93	NM_001245099
il10	R: TCCTGCATTGGACGATCTCT	105	93	NM_001245099
rpl13a	F: GGACAAGCTGCACTGGAGAG	113	100	XM_021574753
rpl13a	R: GTGGGCTTCAGACGGACAAT	113	100	XM_021574753
tnfα	F: CACACTGGGCTCTTCTTCGT	155	98	NM_001124374
tnfα	R: CAAACTGACCTTACCCCGCT	155	98	NM_001124357
ucn2a	F: CATGCGTATGTGTCTGAGCC	61	101	CDQ61544
ucn2a	R: TCTTCTCCCATCCTTTGGCAC	61	101	CDQ61544
ucn2b	F: ACTCCTAAGTTGTGACATTGGA	68	99	XM_021570508
ucn2b	R: CCAGACACAGGCTTAGATG	68	99	XM_021570508
ucn3	F: GCACGGAACTTGGACATCAT	91	100	XM_021596023
ucn3	R: GTCTGAGACAGAGGCTGGAG	91	100	XM_036945073
uts1a	F: GTTGCTGAAGGCTGGTGATA	82	100	NM_001124343
uts1a	R: CAACCGGGGATTTCTTTG	82	100	NM_001124343
uts1b	F: GGCGACGCAGTTTCCTAC	60	99	XM_021612039
uts1b	R: GGGATTTCTCTGCAAATAACG	60	99	XM_021612039

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Abbreviations: crf, corticotropin‐releasing factor; crfbp, corticotropin‐releasing factor binding protein; crfr, corticotropin‐releasing factor receptor; c3, complement 3; ef1α, elongation factor 1α; ifnγ, interferon γ; il, interleukin; rpl13a, ribosomal protein L13a; tnfα, tumor necrotic factor α; ucn, urocortin; uts, urotensin.

2.3. Experimental Design

2.3.1. Experiment 1: Immune Challenge

Eighty‐four sexually immature trout were used for this experiment (mass = 271 ± 6 g, fork length = 27.8 ± 0.2 cm). Fish were lightly anesthetized using MS‐222 (70 mg L⁻¹, Syndel; Vancouver, BC, Canada), weighed, and haphazardly transferred into one of eight experimental tanks (N = 10–11 fish per tank; N = 1 tank per experimental group). Fish were held in 0.6 m diameter fiberglass tanks (~200 L) containing an air stone. All tanks were supplied with flow‐through well water maintained at 12°C and were kept on a 12 h light, 12 h dark photoperiod regime. Each tank was fed 1% of the average cumulative body weight in each tank daily and were held under these conditions for 2 weeks. Following this acclimation period, fish were anesthetized using MS‐222 (100 mg L⁻¹) and injected intraperitoneally with 250 μL of either an inactivated vaccine solution or filter‐sterilized saline (0.9% NaCl). Food was withheld for 24 h prior to injection. The vaccine (ICTHIOVAC VR, HIPRA; Amer, Girona, Spain) contained formalin‐killed Vibrio anguillarum (serotypes O1, O2α and O2β) and has previously been shown to cause an immune response in rainbow trout [49, 50]. After either 6, 24, 72, or 168 h post‐injection, groups of vaccine‐ and saline‐injected fish were killed via terminal anesthesia using 2‐phenoxyethanol (0.2%, Sigma‐Aldrich; Oakville, ON, Canada) at 1400 h. Fork length and mass were recorded, and blood was collected from the caudal vasculature using a 1 mL ammonium heparinized syringe. A portion of the blood was placed on ice to facilitate the evaluation of blood leukocyte abundance and respiratory burst activity (see Section 2.4.1), and the rest was centrifuged at 9000 g for 5 min. After this, plasma was collected, frozen on dry ice, and stored at −80°C for later determination of cortisol levels (see Section 2.4.2). The intestinal tract was removed from each fish, flushed of its contents, and regionally dissected as previously described [51]. Specifically, we designated the middle intestine as the segment between the final pyloric caeca and the ileorectal sphincter, and the posterior intestine as the segment posterior to the ileorectal sphincter. These intestine regions, as well as the second gill arch on the right side of each fish, were frozen on dry ice and stored at −80°C to be used for later evaluation of immune‐related transcripts and transcripts in the CRF system (see Section 2.5). Food was withheld throughout the experiment to avoid potential confounds associated with the inhibition of appetite due to vaccination [47].

2.3.2. Experiment 2: Osmotic Challenge

Fifty‐eight trout were used for this experiment (mass = 544 ± 14 g, fork length = 35.0 ± 0.3 cm). Groups of 10 fish were haphazardly placed into recirculating tanks containing either freshwater or seawater (35 ppt, Instant Ocean Sea Salt; Blacksburg, VA, USA). All tanks contained ~2000 L of water at 12°C and were on a 12 h light, 12 h dark photoperiod regime. Additionally, all tanks were continuously aerated with an air stone and equipped with both particle and biological filtration, as well as UV sterilization. Each group of fish was sampled 24, 72, or 168 h following transfer (N = 1 tank per experimental group). Two fish were removed from the 168 h FW‐to‐SW transferred group prior to the end of the experiment resulting in N = 8 for this group. Plasma (cortisol and osmolality; see Section 2.4.2), gill, and intestine segments (CRF system transcript abundance; see Section 2.5) were collected as described above. As in Experiment (Exp) 1, food was withheld throughout the experiment because salmonids naturally suppress food intake during seawater acclimation [46, 48, 52, 53].

2.3.3. Experiment 3: Effects of Fasting and Refeeding on the Intestine

Forty‐nine trout were used for this experiment (mass = 750 ± 19 g, fork length = 37.9 ± 0.3 cm; N = 27 females and 22 males) and most (> 80%) were still juvenile. Fish were lightly anesthetized using MS‐222 (70 mg L⁻¹), weighed, and haphazardly transferred into the experimental tanks (N = 7 fish per tank; N = 1 tank per timepoint). Fish were held in 1.2 m diameter fiberglass tanks (~500 L) that were supplied with aerated, flow‐through well water. Tanks were maintained at 12°C and kept on a 12 h light, 12 h dark photoperiod regime. For 2 weeks prior to the start of fasting, fish were fed 1.5% of the average cumulative body weight in each tank at 1100 h every day (a pilot study determined this to be the maximum amount that fish would fully consume daily). Following this acclimation period, all fish in one tank were immediately sacrificed (Control). Fish in the other six tanks were sampled following either 24, 72, or 168 h of fasting, or 24, 72, or 168 h after feeding had resumed following a 168 h fast. All treatment groups were terminally anesthetized at 1200 h (1 h after feeding for non‐fasted groups), and plasma (cortisol; see Section 2.4.2) and intestine (CRF system transcript abundance; see Section 2.5) were collected as described above.

2.4. Physiological Parameters

2.4.1. Immune Indicators

For Exp 1, we evaluated several immune‐related variables to confirm that our vaccination protocol was effective in eliciting an immune response at both whole animal (leukocyte counts and respiratory burst in the blood; see below) and tissue levels (transcript abundance of immune‐related genes in each tissue of interest; see Section 2.5).

The total proportion of circulating leukocytes—as well as separate subpopulations of lymphocytes/thrombocytes (called “lymphocytes”) and neutrophil/monocytes (called “neutrophils”)—was quantified using flow cytometry according to the protocol of Inoue et al. [54]. Briefly, 10 μL of whole blood was diluted into 1986 μL of Hank's balanced salt solution (HBSS; Item #H8264, Sigma‐Aldrich) and 4 μL of ethanol containing 500 μg mL⁻¹ DiOC₆ (3,3′‐dihexyloxacarbocyanine iodide; Item #318426, Sigma‐Aldrich). After incubating at room temperature for 10 min in the dark with shaking (100 rpm), samples were filtered with a Falcon cell strainer (35 μm, Corning; Glendale, AZ, USA) and loaded into a FACSMelody cell sorter (RRID:SCR_023209; BD Bioscience; Mississauga, ON, Canada). All samples were agitated at 200 rpm during analysis, and an event rate of ~6000 events s⁻¹ was maintained until 1 million events were recorded. Voltage thresholds were set at 15, 49, and 32 V for forward scatter, side scatter, and fluorescence photomultiplier tubes, respectively. Leukocyte subpopulations (see above) were identified within the FACSChorus software (BD Biosciences) based on differences in forward scatter, side scatter, and fluorescence intensities, as previously described [54].

The respiratory burst activity of leukocytes in the blood was measured as previously described [55]. Briefly, white polystyrene microplates (Corning) were prepared with four wells containing 75 μL of whole blood from each fish (three stimulated and one unstimulated). Stimulated wells received 85 μL of HBSS, 40 μL of luminol in HBSS (10 mM; Item #A8511, Sigma‐Aldrich), and 100 μL of zymosan A in HBSS (20 mg mL⁻¹; Item #Z4250, Sigma‐Aldrich). Unstimulated wells received the same reagents except that 100 μL of HBSS was added in lieu of zymosan solution. The chemiluminescent signal in each well was measured every 3 min over 150 min using an LMax II³⁸⁴ luminometer (Molecular Devices; San Jose, CA, USA). We recorded the integral of the signal over the entire duration and, to account for background signal, the value of the unstimulated well was subtracted from the average value of the three stimulated wells for each fish (intra‐assay variation was 8.8% CV).

2.4.2. Plasma Cortisol and Osmolality

Plasma cortisol levels were measured using a previously validated, commercially available enzyme‐linked immunosorbent assay (ELISA; Item #402710, Neogen; Lexington, KY, USA) for fish from all experiments. Values of intra‐ and inter‐assay variation were 9.3% and 4.5% CV, respectively. Additionally, plasma osmolality values were determined in duplicate using a vapor pressure osmometer (Vapro 5520, Wescor; Logan, UT, USA) for fish from Exp 2, which had an intra‐assay variation of 0.8% CV.

2.5. RNA Isolation and qPCR

Gill and intestine (both middle and posterior regions) samples were ground on dry ice with a mortar and pestle, then homogenized in Ribozol reagent (VWR International; Mississauga, ON, Canada) using a Precellys Evolution tissue homogenizer (Bertin Instruments; Montigny‐le‐Bretonneux, France). Total RNA was extracted following the manufacturer's protocol, after which its quantity and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific; Mississauga, ON, Canada). We then treated 1 μg of RNA with DNase (DNase 1; Thermo Scientific) and reverse‐transcribed cDNA using a high‐capacity Applied Biosystems cDNA reverse transcription kit (Thermo Scientific). We performed qPCR using a CFX96 system (RRID:SCR_018064; BioRad; Hercules, CA, USA) with SYBR green (SsoAdvanced Universal; BioRad) and gene‐specific primers (Table 1). All samples were run in duplicate, and negative controls, including no‐template controls (where cDNA was replaced with water) and no reverse transcriptase controls (where RNA reverse transcriptase was replaced with water during cDNA synthesis) were included. Each reaction contained a total of 20 μL, which consisted of 10 μL of SYBR green, 5 μL of combined forward and reverse primers (0.2 μM [final]), and 5 μL of 10× diluted cDNA. Cycling parameters included a 30 s activation step at 95°C, followed by 40 cycles consisting of a 3 s denaturation step at 95°C and a combined 30 s annealing and extension step at 60°C. Melt curve analysis was conducted at the end of each run to confirm the specificity of each reaction. To account for differences in amplification efficiency, standard curves were constructed for each gene using serial dilutions (4×) of pooled cDNA. Input values for each gene were obtained by fitting the average threshold cycle value to the antilog of the gene‐specific standard curve, thereby correcting for differences in primer amplification efficiency. To correct for minor variations in template input and transcriptional efficiency, we measured transcript abundance of elongation factor 1 α (ef1α) and ribosomal protein L13a (rpl13a) as reference genes, which were both stable across groups in all experiments. Data were normalized using the geometric mean of these reference genes and are expressed relative to the mean value of the 6 h saline group in Exp 1, the 24 h FW group in Exp 2, and the fed group in Exp 3.

2.6. Statistical Analysis

Statistical analyses were performed using R (version 4.4.0, RRID:SCR_001905). All data are presented as means ±1 standard error of the mean (SEM), and a significance level (α) of 0.05 was used for all tests. Statistical outliers (maximum of 2 per group) were excluded based on a 2× interquartile range threshold. When data did not meet the assumptions of normality and/or equal variance, data were transformed to improve model fit. Data from each time course series was analyzed using ANOVAs that included either only time as a factor (Exp 3) or time, treatment, and their interaction term as factors (Exp 1 and 2). All models were fit using the lm function in the “lme4” package (RRID:SCR_015654; [56]) and overall differences were determined using the Anova function in the “car” package (RRID:SCR_022137). When significant differences were detected, Tukey's HSD post hoc analysis was performed using the emmeans function in the “emmeans” package (RRID:SCR_018734; [57]). Sex was not included in our analyses because fish were either sexually immature (Exps 1 and 2) or preliminary analyses revealed no effects of sex (Exp 3). The major findings of our analyses are described in the results section below; however, a detailed description of all nonsignificant statistical results can be found in the electronic Supporting Information (Tables S1–S3).

3. Results

3.1. Tissue Distribution

In general, transcript abundance of binding proteins and receptors was far more abundant in the gills and both regions of the intestine compared to most ligands (Figure 1). While undetectable in either region of the intestine, crfr2a was the most abundant receptor in the gills. Similarly, crfr1a and crfr1b were both ~5× more abundant in the gills than in either the Mid. Int. or Post. Int. Levels of crfr2b were twice as high in the Post. Int. compared to levels in either the gills or Mid. Int. For binding proteins, crfbp1 was the most abundant component expressed in the gills and was ~30× and ~60× more abundant there than in the Mid. Int. and Post. Int., respectively. In contrast, crfbp2 was the most abundant component in the intestine, with ~60× and ~30× greater abundance in the Mid. Int. and Post. Int., respectively, compared to the gills. Lastly, ucn3 was ~4× more abundant in the gills than in the intestine, crfa2 was ~5× more abundant in the intestine than in the gills, and crfa1 levels were low but detectable in all tissues. In contrast, we only detected ucn2b in the intestine, crfb1 and uts1a in the gills, and none of crfb2, uts1b, or ucn2a were detectable in any of the examined tissues.

Relative abundance of individual ligands, binding proteins, and receptors of the corticotropin‐releasing factor system in the gills, middle intestine (Mid. Int.), and posterior intestine (Post. Int.) of rainbow trout ( *Oncorhynchus mykiss* ). Bars represent mean abundance (±SEM) of each component relative to *crfa2* in the gills (the component with the lowest detectable levels). Each point (N = 3) represents an individual value from an unstressed fish. Components that were not detected in any of the three tissues are indicated with nd (not detected), and the ∅ symbol indicates instances where components were only undetectable in some tissues. Note that data are plotted on a Log10 scale for visualization purposes. *crf*, corticotropin‐releasing factor; *crfbp*, corticotropin‐releasing factor binding protein; *crfr*, corticotropin‐releasing factor receptor; *ucn*, urocortin; *uts*, urotensin.

3.2. Experiment 1: Immune Challenge

Vaccination caused total circulating leukocytes to decrease by 50% at 24 h (Figure 2A; p_treatment = 0.63, p_time < 0.001, p_interaction < 0.001), which was driven by a concurrent 50% reduction in lymphocyte counts at 24 h (Figure 2B; p_treatment = 0.02, p_time < 0.001, p_interaction < 0.001). In contrast, blood neutrophil counts in vaccinated fish were ~2.3 and ~1.5× higher at 72 h and 168 h (Figure 2C; p_treatment < 0.001, p_time < 0.001, p_interaction < 0.001), respectively. Similarly, respiratory burst activity in the blood was ~2.5× and ~1.5× greater in vaccinated fish at 72 h and 168 h (Figure 2D; p_treatment = 0.03, p_time < 0.001, p_interaction = 0.04), respectively. Plasma cortisol levels were ~35% higher in vaccinated fish across all time points and ~3× higher at 6 h compared to all other time points in both groups (Table 2); however, plasma cortisol levels were generally low in all fish (< 10 ng mL⁻¹). Consistent with the observed immunological responses in the blood, transcript abundance of immune‐related genes further confirmed that all examined tissues mounted a robust immune response (Table 3). Peak activation of proinflammatory cytokines (il1b, ifnγ, and tnfα at 6–24 h) was followed by the elevation of a key anti‐inflammatory cytokine (il10 at 24 h), as well as a delayed and prolonged activation of the complement system (c3 at 24–168 h). Transcriptional responses of all immune‐related genes were greater in both regions of the intestine compared to the gills.

Time‐dependent effects of saline or vaccine treatment on the percentage of leukocytes (A), lymphocytes (B), and neutrophils (C), as well as respiratory burst (D) in the blood of rainbow trout ( *Oncorhynchus mykiss* ). Significant differences (p < 0.05; two‐way ANOVA) are depicted using either letters (across time; uppercase = within vaccine group, lowercase = within saline group) or asterisks (between groups within a timepoint). Values are represented as means ± SEM, and individual data points are shown (N = 9–11).

TABLE 2.

Time‐dependent effects of vaccination (Exp 1), seawater transfer (Exp 2), and feeding state (Exp 3) on cortisol (ng mL⁻¹; all Exp) and osmolality (mOsm kg⁻¹; only Exp 2) in the plasma of rainbow trout ( Oncorhynchus mykiss ). Data are presented as means ± SEM (N = 7–11 per group). Significant effects are indicated with bold (p < 0.05). Differences as determined using post hoc analysis are depicted using either letters (time effects within a treatment, overall treatment effects, or overall time effects) or asterisks (treatment effect within a timepoint). Significant differences were not detected during post hoc analysis of plasma cortisol levels for Exp 3. Note that plasma cortisol and osmolality values from Exp 1 and 2 have previously been reported [32, 58].

Variable	Exp	Treatment	Sampling Time					F	p
Plasma Cortisol			6 h ^B	24 h ^A	72 h ^A	168 h ^A
	1	Saline^A	7.4 ± 1.6	3.1 ± 0.9	1.1 ± 0.3	4.6 ± 0.7	Treatment	6.32	0.01
		Vaccine^B	11.3 ± 1.3	4.4 ± 1.0	3.2 ± 0.8	3.3 ± 0.5	Time	19.32	< 0.001
							Interaction	2.63	0.06
				24 h	72 h	168 h
	2	FW‐to‐FW		12.9 ± 3.5^a	6.4 ± 3.2^b	10.4 ± 2.7^a	Treatment	39.2	< 0.001
		FW‐to‐SW		36.4 ± 7.8^AB*	105.9 ± 19.9^B*	14.9 ± 4.5^A	Time	1.9	0.16
							Interaction	12.3	< 0.001
			Fed	24 h	72 h	168 h
	3	Fasted	12.0 ± 2.1	12.7 ± 3.0	9.1 ± 1.0	10.9 ± 0.8	Treatment	3.71	0.005
		Fasted & Refed		5.4 ± 1.0	4.7 ± 0.6	7.9 ± 2.2
Plasma Osmolality				24 h	72 h	168 h
	2	FW‐to‐FW^A		293 ± 1	294 ± 1	295 ± 2	Treatment	177.8	< 0.001
		FW‐to‐SW^B		359 ± 4	385 ± 7	349 ± 12	Time	2.8	0.07
							Interaction	2.3	0.11

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TABLE 3.

Time‐dependent effects of saline or vaccine treatment on immune‐related genes in the gills, middle intestine (Mid. Int.), and posterior intestine (Post. Int.) of rainbow trout ( Oncorhynchus mykiss ). Data are presented as means ± SEM (N = 8–11 per group) and are expressed relative to the saline group at 6 h. Significant effects are indicated with bold (p < 0.05). Differences as determined using post hoc analysis are depicted using either letters (time effects within a treatment) or asterisks (treatment effect within a time point).

Tissue	Gene	Treatment	Sampling time					F	p
Tissue	Gene	Treatment	6 h	24 h	72 h	168 h		F	p
Gills	il1b	Saline	1.00 ± 0.09	0.93 ± 0.08	0.88 ± 0.05	1.08 ± 0.07	Treatment	678.9	< 0.001
		Vaccine	36.53 ± 2.90^A*	6.66 ± 0.93^B*	1.90 ± 0.11^C*	1.19 ± 0.07^D	Time	148.3	< 0.001
							Interaction	155.3	< 0.001
	il10	Saline	1.00 ± 0.16^a	0.49 ± 0.07^b	1.04 ± 0.13^a	0.42 ± 0.03^b	Treatment	11.0	0.001
		Vaccine	0.75 ± 0.08^A	2.42 ± 0.31^B*	1.15 ± 0.16^A	0.30 ± 0.03^C*	Time	38.9	< 0.001
							Interaction	30.1	< 0.001
	ifny	Saline	1.00 ± 0.11	0.84 ± 0.06	1.09 ± 0.10	0.76 ± 0.10	Treatment	30.1	< 0.001
		Vaccine	1.01 ± 0.07^A	3.35 ± 0.65^B*	1.76 ± 0.23^C*	0.76 ± 0.07^A	Time	17.9	< 0.001
							Interaction	12.9	< 0.001
	tnfα	Saline	1.00 ± 0.06^ab	0.90 ± 0.02^ab	0.81 ± 0.04^a	1.02 ± 0.04^b	Treatment	142.6	< 0.001
		Vaccine	4.74 ± 0.34^A*	1.47 ± 0.15^B*	0.99 ± 0.06^C*	0.87 ± 0.04^C	Time	89.9	< 0.001
							Interaction	73.7	< 0.001
	c3	Saline	1.00 ± 0.08	1.03 ± 0.10	0.88 ± 0.05	1.00 ± 0.08	Treatment	105.4	< 0.001
		Vaccine	1.22 ± 0.09^A	2.96 ± 0.48^B*	3.08 ± 0.41^B*	1.29 ± 0.05^A*	Time	11.5	< 0.001
							Interaction	15.1	< 0.001
Mid. Int.	il1b	Saline	1.00 ± 0.17^ab	1.42 ± 0.27^a	0.48 ± 0.04^b	0.47 ± 0.04^b	Treatment	696.7	< 0.001
		Vaccine	278.49 ± 39.45^A*	101.06 ± 39.02^B*	18.68 ± 2.70^C*	2.12 ± 0.53^D*	Time	87.8	< 0.001
							Interaction	44.5	< 0.001
	il10	Saline	1.00 ± 0.16	1.12 ± 0.20	1.03 ± 0.21	0.77 ± 0.07	Treatment	210.7	< 0.001
		Vaccine	11.48 ± 2.69^A*	24.62 ± 6.05^B*	6.90 ± 1.15^A*	1.22 ± 0.14^C	Time	23.9	< 0.001
							Interaction	16.6	< 0.001
	ifny	Saline	1.00 ± 0.10	1.54 ± 0.14	1.48 ± 0.13	1.56 ± 0.16	Treatment	224.7	< 0.001
		Vaccine	5.60 ± 1.56^A*	16.57 ± 1.70^B*	10.60 ± 1.68^B*	3.52 ± 0.59^A*	Time	15.7	< 0.001
							Interaction	10.6	< 0.001
	tnfα	Saline	1.00 ± 0.11^a	1.81 ± 0.17^b	1.35 ± 0.12^ab	1.42 ± 0.11^ab	Treatment	234.7	< 0.001
		Vaccine	9.71 ± 0.72^AB*	17.29 ± 4.14^B*	6.91 ± 1.10^A*	2.05 ± 0.25^C	Time	20.1	< 0.001
							Interaction	19.6	< 0.001
	c3	Saline	1.00 ± 0.16^a	1.57 ± 0.32^ab	1.90 ± 0.20^b	1.72 ± 0.29^ab	Treatment	618.6	< 0.001
		Vaccine	10.20 ± 2.10^A*	36.67 ± 8.09^B*	139.30 ± 18.64^C*	40.73 ± 6.17^B*	Time	28.6	< 0.001
							Interaction	9.8	< 0.001
Post. Int.	il1b	Saline	1.00 ± 0.14	0.95 ± 0.08	1.43 ± 0.20	0.97 ± 0.10	Treatment	581.0	< 0.001
		Vaccine	181.55 ± 25.83^A*	78.84 ± 26.38^B*	28.57 ± 5.65^C*	2.40 ± 0.41^D*	Time	48.7	< 0.001
							Interaction	48.4	< 0.001
	il10	Saline	1.00 ± 0.15^a	2.89 ± 0.54^b	1.88 ± 0.34^ab	1.30 ± 0.32^a	Treatment	84.3	< 0.001
		Vaccine	7.14 ± 1.47^A*	24.12 ± 4.42^B*	5.27 ± 0.94^A*	1.02 ± 0.10^C	Time	36.0	< 0.001
							Interaction	14.0	< 0.001
	ifny	Saline	1.00 ± 0.13^a	2.07 ± 0.33^b	2.01 ± 0.28^b	0.77 ± 0.08^a	Treatment	126.9	< 0.001
		Vaccine	2.96 ± 0.35^A*	13.21 ± 2.22^B*	5.06 ± 0.70^A*	1.41 ± 0.10^C*	Time	44.0	< 0.001
							Interaction	6.5	< 0.001
	tnfα	Saline	1.00 ± 0.10	1.52 ± 0.12	1.33 ± 0.10	1.25 ± 0.14	Treatment	197.7	< 0.001
		Vaccine	9.79 ± 1.23^AB*	15.95 ± 3.43^B*	6.36 ± 1.16^A*	1.69 ± 0.22^C	Time	20.5	< 0.001
							Interaction	17.5	< 0.001
	c3	Saline	1.00 ± 0.18^a	1.67 ± 0.27^ab	2.38 ± 0.35^b	1.59 ± 0.21^ab	Interaction	455.4	< 0.001
		Vaccine	8.54 ± 2.12^A*	74.33 ± 20.24^B*	93.57 ± 12.61^B*	46.70 ± 6.79^B*	Treatment	25.2	< 0.001
							Time	7.2	< 0.001

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Abbreviations: c3, complement 3; ifny, interferon gamma; il1b, interleukin 1 beta; il10, interleukin 10; tnfα, tumor necrosis factor alpha.

In the Mid. Int., levels of crfbp1 (Figure 3A) and crfbp2 (Figure 3B) were both elevated in vaccinated fish compared to saline‐treated controls. Specifically, crfbp1 was ~2.5× and ~2× higher at 24 and 72 h (p_treatment < 0.001, p_time < 0.001, p_interaction < 0.001), respectively, while crfbp2 in vaccinated fish was ~25% higher overall (p_treatment = 0.04, p_time = 0.41, p_interaction = 0.32). In contrast, levels of crfr1b (Figure 3C; p_treatment = 0.03, p_time < 0.001, p_interaction = 0.19) and crfa2 (Figure 3D; p_treatment = 0.002, p_time < 0.001, p_interaction = 0.59) were lower in vaccinated fish compared to saline‐treated fish. This effect was greatest at 24 h for crfr1b—with levels in vaccinated fish being 25% those of saline‐treated fish—but levels of crfa2 were ~20% lower in vaccinated fish across all time points. Additionally, the abundance of crfr1b and crfa2 was 2–3‐fold higher overall at 24, 72, and 168 h compared to 6 h. No vaccine‐related changes were observed for crfr2b or ucn2b (Supp. Table 1).

Time‐dependent effects of saline or vaccine treatment on transcript abundance of corticotropin‐releasing factor (CRF) binding protein 1 (*crfbp1*; A), CRF binding protein 2 (*crfbp2*; B), CRF receptor 1b (*crfr1b*; C), and CRFa2 (*crfa2*; D) in the middle intestine (Mid. Int.) of rainbow trout ( *Oncorhynchus mykiss* ). Significant differences (p < 0.05; two‐way ANOVA) are depicted using either letters (across time; uppercase = within vaccine group, underlined uppercase = overall time effect), filled oversized squares (between groups across all timepoints) or asterisks (between groups within a timepoint). Data are expressed relative to saline‐injected fish at 6 h. Values are represented as means ± SEM, and individual data points are shown (N = 8–11).

In the Post. Int., changes in crfbp1 (Figure 4A) and crfr1b (Figure 4B) mirrored those observed in the Mid. Int. Specifically, levels of crfbp1 in vaccinated fish were ~2.5× higher than those of saline‐treated fish at both 24 and 72 h (p_treatment < 0.001, p_time < 0.001, p_interaction = 0.006), and levels of crfr1b were ~70% lower than those of saline‐treated fish at 24 h (p_treatment = 0.08, p_time = 0.21, p_interaction = 0.01). While unaffected by vaccination, levels of crfa2 (Figure 4C; p_treatment = 0.62, p_time < 0.001, p_interaction = 0.07) gradually increased across the duration of the experiment with levels at 168 h being twice those at 6 h. No vaccine‐related changes were observed for crfbp2, crfr2b, or ucn2b (Table S1).

In contrast to the intestine, the only vaccine‐related change observed in the gills was lower crfr2a levels in vaccinated fish compared to saline‐treated fish at 72 h (Table S1). However, this difference was primarily driven by a transient reduction in the saline group at 72 h and not by altered crfr2a levels in vaccinated fish. Similarly, no vaccine‐related changes were observed for crfbp1, crfr1a, crfr1b, crfr2b, or ucn3 (Table S1).

3.3. Experiment 2: Osmotic Challenge

As previously reported [32], plasma cortisol and osmolality values (Table 2) were both elevated in SW versus FW fish. Plasma cortisol levels were ~3× and ~17× higher in SW fish at 24 and 72 h, respectively, whereas osmolality was ~25% higher in SW fish across the duration of the experiment.

In the Mid. Int., time‐dependent increases in all CRF components were observed in SW fish. Levels of crfbp1 (Figure 5A) transiently increased 2.5‐fold in SW fish at 72 h (p_treatment = 0.04, p_time = 0.86, p_interaction < 0.001), whereas levels of crfbp2 (Figure 5B; p_treatment = 0.70, p_time < 0.001, p_interaction = 0.001), crfr1b (Figure 5C; p_treatment = 0.20, p_time < 0.001, p_interaction < 0.001) and crfr2b (Figure 5D; p_treatment = 0.08, p_time < 0.001, p_interaction < 0.001) in SW fish were ~50% those of FW fish at 24 h, but had increased to levels ~2‐fold higher than FW fish by 168 h. Levels of crfa2 (Figure 5E; p_treatment = 0.20, p_time < 0.001, p_interaction < 0.001) and ucn2b (Figure 5F; p_treatment = 0.03, p_time = 0.003, p_interaction = 0.32) were also higher in SW fish compared to FW fish—especially at 72 (~3× higher) and 168 h (~60% higher). Additionally, transcript levels of both peptides increased 2–3‐fold over the course of the experiment.

Time‐dependent effects of transfer from freshwater‐to‐freshwater (FW) or freshwater‐to‐seawater (SW) on transcript abundance of corticotropin‐releasing factor (CRF) binding protein 1 (*crfbp1*; A), CRF binding protein 2 (*crfbp2*; B), CRF receptors 1b (*crfr1b*; C) and 2b (*crfr2b*; D), CRFa2 (*crfa2*; E), and urocortin 2b (*ucn2b*; F) in the middle intestine (Mid. Int.) of rainbow trout ( *Oncorhynchus mykiss* ). Significant differences (p < 0.05; two‐way ANOVA) are depicted using either letters (across time; uppercase = within SW, lowercase = within FW, underlined uppercase = overall time effect), filled oversized squares (between groups across all timepoints) or asterisks (between groups within a timepoint). Data are expressed relative to FW fish at 24 h. Values are represented as means ± SEM, and individual data points are shown (N = 7–10).

In the Post. Int., levels of crfbp1 (Figure 6A) increased ~3‐fold across time in FW fish but not in SW fish (p_treatment = 0.27, p_time = 0.10, p_interaction = 0.046). Similar increases of ~2–3‐fold across the duration of the experiment were observed for crfbp2 (Figure 6B; p_treatment = 0.85, p_time = 0.03, p_interaction = 0.68), crfr1b (Figure 6C; p_treatment = 0.71, p_time = 0.06, p_interaction = 0.99), crfa2 (Figure 6D; p_treatment = 0.48, p_time = 0.04, p_interaction = 0.08), and ucn2b (Figure 6E; p_treatment = 0.97, p_time = 0.03, p_interaction = 0.85); however, these changes were consistent between FW and SW fish. No differences were detected for crfr2b (Table S2).

In the gills, levels of crfbp1 (Figure 7A), crfr1a (Figure 7B), crfr1b (Figure 7C), and crfr2a (Figure 7D) were all lower in SW fish. Time‐dependent reductions of ~30% were detected in SW fish for crfbp1 (p_treatment = 0.44, p_time = 0.02, p_interaction = 0.008) and crfr1b (p_treatment = 0.10, p_time = 0.74, p_interaction = 0.04) at 168 h and 72 h, respectively. Levels of crfr1a (p_treatment = 0.003, p_time = 0.65, p_interaction = 0.77) and crfr2a (p_treatment < 0.001, p_time = 0.46, p_interaction = 0.20) in SW fish were ~30% and ~40% lower, respectively, across all timepoints. No differences in crfr2b or ucn3 were detected (Table S2).

3.4. Experiment 3: Fasting and Re‐Feeding

In the Mid. Int., abundance of CRF system components—specifically, crfbp1 (Figure 8A; p = 0.03), crfbp2 (Figure 8B; p = 0.03), crfr1b (Figure 8C; p = 0.01), and crfr2b (Figure 8D; p < 0.001)—generally decreased (by up to 50%) over the course of the 7d fast. Transcript levels gradually returned to levels consistent with fed animals once feeding resumed. In the Post. Int., levels of crfbp2 (Figure 9A; p < 0.001), crfr1b (Figure 9B; p = 0.009), crfr2b (Figure 9C; p = 0.02), and crfa2 (Figure 9D; p = 0.02) all increased ~2–4‐fold during the 7d fast. Interestingly, while abundance of these genes all returned to levels consistent with fed animals by the end of the 7d re‐feeding period, levels of crfbp2, crfr1b, and crfr2b initially increased ~2–3‐fold prior to this decline. No changes were observed in crfa2 and ucn2b in the Mid. Int. or ucn2b and crfbp1 in the Post. Int. (Table S3).

Time‐dependent effects of fasting and re‐feeding on transcript abundance of corticotropin‐releasing factor (CRF) binding proteins 1 (*crfbp1*; A) and 2 (*crfbp2*; B), and CRF receptors 1b (*crfr1b*; C) and 2b (*crfr2b*; D) in the middle intestine (Mid. Int.) of rainbow trout ( *Oncorhynchus mykiss* ). Significant differences (p < 0.05; one‐way ANOVA) are depicted using letters; except in Panel A where the p value is reported because no significant post hoc comparisons were detected. Data are expressed relative to control fish that were fed 1 h prior to sampling. Values are represented as means ± SEM and individual data points are shown (N = 6–7).

Time‐dependent effects of fasting and re‐feeding on transcript abundance of corticotropin‐releasing factor (CRF) binding protein 2 (*crfbp2*; A), CRF receptors 1b (*crfr1b*; B) and 2b (*crfr2b*; C), and CRFa2 (*crfa2*; D) in the posterior intestine (Post. Int.) of rainbow trout ( *Oncorhynchus mykiss* ). Significant differences (p < 0.05; one‐way ANOVA) are depicted using letters. Data are expressed relative to control fish that were fed 1 h prior to sampling. Values are represented as means ± SEM and individual data points are shown (N = 5–7).

While a significant difference in plasma cortisol levels was observed during the fasting‐refeeding experiment (Table 2), no differences were detected during subsequent post hoc analysis, and cortisol levels were generally low across all groups (< 15 ng mL⁻¹).

4. Discussion

While neuroendocrine functions for the CRF system in the brain (e.g., regulation of the HPA/HPI axis) are well established [5, 20], stress‐related roles for the CRF system outside of the brain are less well understood, especially in nonmammalian vertebrates. In the current study, we first established that most components of the CRF system were present in both the gills and intestine of rainbow trout; however, we were unable to detect crfb2, uts1b, or ucn2a in either tissue, and uts1a (gills), crfb1 (gills), and ucn2b (intestine) were only detectable in one of these tissues. These findings are consistent with our previous studies of the peripheral CRF system in salmonids (e.g., spleen, intestine, and gills [33, 34, 58]), which collectively suggest that only a subset of CRF ligands (e.g., CRFa and UCN3) have para/autocrine functions within peripheral tissues. Instead, it seems likely that circulating CRF peptides originating from the caudal neurosecretory system—especially CRFb and UTS1 [32, 59]—also contribute to CRF system regulation in these tissues. This hypothesis is further supported by the high levels of CRFBP measured in the gills and intestine. Since CRFBP has high affinity for CRFb and UTS1 (but not UCN3 [60, 61]), CRFBP may determine the relative responsiveness of these tissues to changes in circulating levels of CRF ligands. Overall, our distribution data support physiological functions for the CRF system in both the gills and intestine of rainbow trout.

To evaluate whether components of the CRF system were regulated in a tissue‐ and stressor‐specific manner, we first characterized time‐dependent responses following an immune challenge (i.e., vaccine administration). Vaccinated fish had higher blood neutrophil counts and respiratory burst activity, indicating that fish mounted a robust immune response. Vaccination was also associated with lower circulating lymphocytes at 24 h, likely reflecting the movement of lymphocytes from the blood to lymphoid organs and the site of vaccination in support of systemic and local immune responses [62, 63, 64]. Consistent with immune responses observed in the blood, we also detected large (~15–300‐fold) increases in immune‐related transcripts across both the middle and posterior portions of the intestine following vaccination. These responses occurred in parallel with transcriptional changes in several CRF system components 24‐72 h post‐vaccination; specifically, an upregulation of CRFBP (especially crfbp1) combined with a downregulation of crfr1b and crfa2. We also found that splenic levels of these same CRF system components displayed similar transcriptional changes [58] suggesting that suppression of CRFR1 signaling in response to vaccination may be widespread across tissues. In mammals, activation of the CRF system—with specific effects mediated by both CRFR1 and CRFR2—causes wide‐ranging proinflammatory effects in the intestine, including increased production of proinflammatory cytokines (e.g., IL6, IL8, and TNFα [21, 22, 23]). While no previous study has evaluated immune contributions of the CRF system in the intestine of fish, Jiang et al. [26] suggested that anti‐inflammatory actions of UTS1 in ayu ( Plecoglossus altivelis ) head kidney leukocytes were caused by UTS1‐mediated inhibition of IL6 signaling pathways. While these findings contrast with mammalian studies, UTS1 treatment also elicited immunosuppressive effects (reduced phagocytotic activity) in cultured spotted snakehead ( Channa punctatus ) spleen monocytes [28]. Our results do not support an anti‐inflammatory role for the intestinal CRF system in trout since the observed reductions in transcript levels of crfr1b and crfa2 coincided with peak production of the anti‐inflammatory cytokine il10, however, it is possible that the observed transcriptional changes in CRF system components facilitate greater phagocytotic activity following vaccination. Indeed, Singh & Rai [28] used receptor‐specific antagonists to show that the anti‐phagocytotic effects associated with UTS1 were mediated by CRFR1 (and not CRFR2), which is consistent with the observed downregulation of intestinal crfr1b at 24 h post‐vaccination. Additionally, van Heijningen et al. [29] reported that mutant zebrafish larvae lacking crfr1 recruited fewer macrophages and more neutrophils following tail fin amputation, offering further evidence that CRFR1 has important immune functions in teleosts. Overall, while our transcriptional results are consistent with CRFR1‐mediated immunoregulatory roles in the intestine of trout, additional work is needed to determine whether these roles vary between species, tissues, cell types, and/or immune components (e.g., innate versus adaptive immune systems).

Unlike in the intestine, we did not detect any transcriptional changes in the gill CRF system following vaccination. As discussed above, since the CRF system generally has proinflammatory effects in mammals, we predicted an acute activation of the CRF system during the initial surge of proinflammatory cytokine production, followed by suppression of CRF system activity as anti‐inflammatory cytokines are activated [22, 65]—the latter of which was observed in the intestine. Indeed, Jiang et al. [26] found that transcript levels of UTS1 acutely increased in the gills of ayu following infection with V. anguillarum (12‐24 h after onset of infection). In contrast, Mazon et al. [27] reported that chronic (3 weeks) infection with the parasite Trypanoplasma borreli caused transcript levels of crfr1 and crfbp in the gills of common carp to decrease by ~60%. These different responses could reflect several factors, including species‐specific regulation of the gill CRF system, the type of immune challenge used (vaccination versus infection with an actual pathogen), and/or whether cortisol levels were concurrently elevated. Similarly, it is possible that the immune challenge used in the current study failed to elicit a strong enough local immune response to affect the gill CRF system, especially since previous in vitro work has shown that the CRF system can influence proinflammatory cytokine systems in Atlantic salmon ( Salmo salar ) gills (e.g., IL23 and IL17 receptors; [34]). While we detected significant transcriptional responses for all immune‐related genes that were measured in the gills, these responses were weaker than those observed in the intestine (current study) or spleen [58], despite similar baseline expression across tissues. However, changes in gill CRF system components were observed by Mazon et al. [27] despite T. borreli infection not being associated with large changes in cytokine expression in carp [66]. Therefore, it is possible that changes in the gill CRF system following parasite infection reflect other actions associated with CRF in the gills (e.g., regulation of angiogenesis and/or endothelial permeability; [34]) and might not have been directly contributing to immunoregulatory functions. Thus, future investigations evaluating how immune challenges of different types and intensities influence the CRF system across tissues and between species will help to determine the extent to which the CRF system serves immune functions in teleosts.

In addition to the immune challenge, we also evaluated how the peripheral CRF system responded to an osmotic stressor. Following SW transfer, fish mounted a cortisol response lasting at least 72 h (but not more than 168 h) and displayed elevated plasma osmolality during the entire 168 h experimental period. Both responses indicate that fish were perturbed by this osmotic stressor [46, 67]; however, the reduction in plasma cortisol levels by 168 h suggests that fish had begun to acclimate by this time. Consistent with the plasma responses observed, we also detected transcriptional changes in several components of the gill CRF system during this period. Most notably, crfr1a and crfr2a were downregulated across all sampling times in SW transferred fish, while levels of crfr1b and crfbp1 were lower in SW transferred fish at 72 and 168 h, respectively. These findings are largely consistent with our previous work in Atlantic salmon, where SW transfer was associated with a general transcriptional downregulation of the CRF system (and vice‐versa when SW‐acclimated salmon were transferred into FW)—especially crfr2a and ucn2a [34]. Indeed, the tissue distribution of CRF system components in rainbow trout (current study) and Atlantic salmon [34] also shares many similarities, especially high expression of crfr2a and crfbp1, suggesting that the role(s) of the CRF system in the gills are likely conserved among salmonids. We have previously suggested that these changes in gill CRF system transcription reflect regulatory contributions of the CRF system toward angiogenesis, endothelial permeability, and/or immune regulation in the gills because in vitro treatment of gill filaments with CRFa affected levels of transcripts related to these physiological processes [34]. However, it is likely that the role of the gill CRF system varies between species since levels of crfr1 and crfb were ~5–15‐fold higher in tilapia ( Oreochromis mossambicus ) 30 days after transfer from FW‐to‐SW [30], contrary to the downregulation of CRFR1 observed here. Our previous work in Atlantic salmon [34] also indicates that the observed downregulation of crfr1a and crfr1b (at least during the first 72 h following SW transfer) could be mediated by elevated cortisol levels. Yet, there also appear to be species‐specific differences in the regulatory effects of corticosteroids on components of the gill CRF system since crfr1 levels were increased when black porgy ( Acanthopagrus schlegelii ) gill filaments were treated in vitro with dexamethasone for 48 h [31]. Regardless, the current data clearly support the hypothesis that the CRF system has osmoregulatory functions in the gills of salmonids.

In contrast to the gills, while transcript levels of several CRF system components in the Mid. Int. were initially downregulated following SW transfer (crfbp2, crfr1b, and crfr2b), the abundance of most CRF system components was elevated by 168 h post‐transfer to SW. These contrasting responses between tissues likely reflect the different osmoregulatory role(s) of the gills versus the intestine in SW. Whereas the gills are responsible for exporting ions out of the body, ions are taken up across the intestine to maintain a favorable gradient facilitating water absorption [37, 38, 39]. However, it is not clear why greater transcription of CRF system components in the intestine of SW‐acclimated fish would be beneficial from an osmoregulatory standpoint since the CRF system generally promotes ion and/or water excretion (or reduces absorption) across taxa and epithelium types [25, 36, 68]. Indeed, activation of CRFR2 in the intestine of FW‐acclimated Atlantic salmon reduced absorption of water, Na⁺, and Cl⁻ [33]. Similarly, Mainoya & Bern [35] reported that UTS1 treatment had inhibitory effects on water, Na⁺, and Cl⁻ uptake in the intestine of FW‐acclimated (but not SW‐acclimated) Mozambique tilapia, suggesting a greater role for the intestinal CRF system in FW. Therefore, it is possible that the observed changes in the CRF system during SW acclimation may indirectly assist with water and/or solute transport by affecting processes related to angiogenesis and/or endothelial permeability in the Mid. Int. [34]. Unlike the Mid. Int., few changes in CRF system transcript levels were observed in the Post. Int. following SW transfer. Instead, transcript levels of several genes (crfbp1, crfbp2, crfr1b, crfa2, and ucn2b) increased across time in both the FW‐ and SW‐transferred groups. Indeed, time‐dependent increases in crfa2 transcript levels were also observed in the Post. Int. during the vaccination experiment. Since fish were fasted during both experiments—replicating natural responses of salmonids following immune and osmotic stressors [46, 47, 48, 52, 53]—we decided to evaluate whether changes in feeding state might explain these responses.

The hypothalamic CRF system is a potent regulator of food intake in fish and other vertebrates [42, 44, 69], but the relationship between food intake and CRF system components located outside of the brain is less clear. Several previous studies have reported the presence of various CRF system components in the teleost intestine [70, 71], but we believe that our data are the first to show that transcript abundance of CRF system components in the intestine can vary with feeding state. Interestingly, responses in the middle versus posterior regions of the intestine exhibited opposing patterns, wherein levels declined in the Mid. Int. and increased in the Post. Int. As previously mentioned, activation of the CRF system reduces absorption of water and ions across the intestine in fish and the colon in mammals [33, 35, 72]. While these effects are mediated by CRFR1 in mammals [25], activation of CRFR2 was primarily responsible for these effects in Atlantic salmon [33]. Interestingly, peripheral treatment with CRF peptides can also delay clearance rates in the stomach and small intestine via a CRFR2‐dependent mechanism in mammals [25]. Thus, transcriptional changes in the CRF system observed in both the middle (reduced abundance, suggesting faster clearance rates) and posterior (increased abundance, suggesting reduced absorption rates) regions of the intestine are consistent with CRF contributing to reductions in intestine functionality and investment during extended fasts. While the functional importance of such CRF‐mediated changes is unclear, they may contribute to fasting‐related changes in nutrient/water absorption, immune regulation, and/or alterations in the microbiome [73, 74, 75]. However, since fasting‐related transcriptional responses in both regions of the intestine were generally different than those following either SW transfer or vaccination, the role of the intestinal CRF system is clearly complex and warrants additional study.

In conclusion, we found that the gill and intestine CRF systems are differentially regulated by osmotic and immune stressors. While transcription of CRF system components in the gills decreased following SW transfer, we observed no transcriptional response in the gill CRF system during an immune stressor. In contrast, transcript levels of most intestine CRF system components decreased in response to an immune challenge but increased in the middle intestine following SW transfer. Lastly, fasting caused region‐specific transcriptional changes in the CRF system within the middle (reduction) and posterior (activation) regions of the intestine. Overall, our findings highlight the breadth and functional diversity of stress‐related roles that the CRF system serves outside of the brain.

Author Contributions

Brett M. Culbert: conceptualization, data curation, formal analysis, investigation, methodology, supervision, validation, visualization, writing – original draft, writing – review and editing. Alexis E. Pulford‐Thorpe: investigation, writing – review and editing. Carol Best: investigation, methodology, validation, writing – review and editing. Nicholas J. Bernier: conceptualization, funding acquisition, investigation, methodology, resources, supervision, writing – review and editing.

Funding

This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant provided to NB (RGPIN‐2022‐03151). BMC was supported by a NSERC Doctoral Canadian Graduate Scholarship (CGS‐D) and an Ontario Graduate Scholarship (OGS). CB was supported by a NSERC postdoctoral scholarship.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Time‐dependent effects of saline or vaccine treatment on transcript abundance of CRF system components in the gills, and middle (Mid. Int.) and posterior (Post. Int.) segments of the intestine in rainbow trout ( Oncorhynchus mykiss ). Data are presented as means ± SEM (N = 8 = 11 per group). Significant effects are indicated with bold (p < 0.05) and differences based on post hoc analysis are indicated using letters (across time within a group), asterisks (between groups within a timepoint) or symbols (overall time effects).

Table S2: Time‐dependent effects of transfer from freshwater‐to‐freshwater (FW) or freshwater‐to‐seawater (SW) on transcript abundance of CRF system components in the gills and posterior intestine (Post. Int.) in rainbow trout ( Oncorhynchus mykiss ). Data are presented as means ± SEM (N = 6 = 10 per group).

Table S3: Time‐dependent effects of fasting and refeeding on transcript abundance of CRF system components in the middle (Mid. Int.) and posterior (Post. Int.) segments of the intestine in rainbow trout ( Oncorhynchus mykiss ). Data are presented as means ± SEM (N = 6–7 per group). Significant effects are indicated with bold (p < 0.05) and differences between groups based on post hoc analysis are indicated using letters. Note that significant differences were not detected during post hoc analysis of crfbp1 in the Mid. Int.

FSB2-40-e72270-s001.pdf^{(302.9KB, pdf)}

Acknowledgments

We thank Matt Cornish, Carolyn Trombley, and Mike Davies for their assistance with fish care and advice regarding experimental setups, as well as Dr. Marcia Chiasson and the staff at the Ontario Aquaculture Research Centre for providing us with the rainbow trout. We also thank Emma Mossington and Shayla Larson for their help during dissections, Sarah Alderman for allowing use of the flow cytometer, and Brian Dixon and Tania Rodríguez‐Ramos for assistance with the respiratory burst assay.

Contributor Information

Brett M. Culbert, Email: brett.m.culbert@gmail.com.

Nicholas J. Bernier, Email: nbernier@uoguelph.ca.

Data Availability Statement

Data are deposited on Mendeley Data (doi: http://doi.org/10.17632/bnr9zp28fp.1).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FSB2-40-e72270-s001.pdf^{(302.9KB, pdf)}

Data Availability Statement

Data are deposited on Mendeley Data (doi: http://doi.org/10.17632/bnr9zp28fp.1).

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Stressor‐ and Tissue‐Specific Regulation of the Corticotropin‐Releasing Factor System Across Epithelial Tissues in Rainbow Trout

Brett M Culbert

Alexis E Pulford‐Thorpe

Carol Best

Nicholas J Bernier

ABSTRACT

1. Introduction

2. Methods

2.1. General Housing

2.2. Evaluating the CRF System of the Gills and Intestine

TABLE 1.

2.3. Experimental Design

2.3.1. Experiment 1: Immune Challenge

2.3.2. Experiment 2: Osmotic Challenge

2.3.3. Experiment 3: Effects of Fasting and Refeeding on the Intestine

2.4. Physiological Parameters

2.4.1. Immune Indicators

2.4.2. Plasma Cortisol and Osmolality

2.5. RNA Isolation and qPCR

2.6. Statistical Analysis

3. Results

3.1. Tissue Distribution

FIGURE 1.

3.2. Experiment 1: Immune Challenge

FIGURE 2.

TABLE 2.

TABLE 3.

FIGURE 3.

FIGURE 4.

3.3. Experiment 2: Osmotic Challenge

FIGURE 5.

FIGURE 6.

FIGURE 7.

3.4. Experiment 3: Fasting and Re‐Feeding

FIGURE 8.

FIGURE 9.

4. Discussion

Author Contributions

Funding

Conflicts of Interest

Supporting information

Acknowledgments

Contributor Information

Data Availability Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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