To provide information on the scale cortisol concentration in common dab, a two-pronged approach was chosen based on (1) field measurements and (2) a laboratory in vivo-study. Methodology for both approaches is described separately below.
Field measurements of scale cortisol concentration
In total, 67 common dabs were caught at various locations in the Belgian part of the North Sea in 2015 (n = 11), 2016 (n = 11), and 2017 (n = 45). More information on the campaigns and capture methods can be found in Vercauteren et al. [32]. Some fish showed an acute or healing skin lesion (n = 40) or skeletal deformity (n = 8). Following euthanasia (benzocaïne, 200 mg L−1), scales were scraped off from the pigmented side of the fish. The retrieved scales were rinsed with distilled water to remove mucus and other potential sources of exogenous glucocorticoids. Subsequently, scales were air-dried after which 50 scales were collected in an Eppendorf tube and frozen (- 20 °C) until analysis of the scale cortisol concentration (SCC) (see further). The sampling was carried out in accordance with the approved guidelines and legislation in force with regard to animal welfare.
Laboratory in vivo study
Animals and housing
Thirty-six clinically healthy common dabs (20.9 ± 2.0 cm; 83.3 ± 27.5 g; age between 3 and 10 years) were caught in the Belgian part of the North Sea by the Research Vessel “Simon Stevin”. All fish (> 18 cm) were placed in a survival tank (1 × 1.2 × 1 m; 640 L) with continuous water renewal. Within four hours after capture, fish were transferred to the Marine Station of Ostend (MSO, Flanders Marine Institute – VLIZ) to large recirculating tanks (diameter 2.6 m, 4000 L) with sand covered bottoms (6 cm layer thickness, 0–2 mm grain size). During the acclimatization period at the MSO, water quality was monitored daily and water was renewed when needed to keep water parameters within preset ranges (pH 8.0 ± 0.5; 32 ± 1 practical salinity unit (PSU); 85 ± 5% oxygen saturation). Temperature was raised in due course (+ 1 °C in 48 h) from 9 ± 1 °C up to 16 ± 1 °C. Fish were fed with fresh whiting (Merlangius merlangus) and, after 10 days, feed was gradually switched to commercial feed (Efico Sigma 862, pellet size 4.5 mm, Biomar SAS, France). After an acclimatization period of three weeks, fish were transported (1 h) to the experimental tanks at the Faculty of Veterinary Medicine (Ghent University) using small transportation boxes (39.4 × 59.8 × 18.6 cm; 30 L) provided with an oxygen tablet (JBL GmbH & Co.KG, Germany).
Upon arrival at the experimental facility, fish were randomly distributed over six circular tanks (diameter 1.1 m; 800 L). The sex ratio in the tanks was similar. Tanks (six fish per tank) were filled with recirculating seawater and a sand layer (6 cm layer thickness, 0-2 mm grain size). Water quality was monitored daily, whereby throughout the experiment, following average values were recorded: 16.68 ± 0.48 °C; pH 8.97 ± 0.26; 31.36 ± 0.67 PSU; 82.17 ± 6.79% dissolved oxygen. Ammonia and nitrite levels never exceeded 0.2 and 1 mg L−1, respectively. Upon renewal of the water, artificial sea water was added (Instant Ocean, Aquarium Systems, USA). Due to presence of natural lighting, a natural day-night rhythm was provided as encountered between June and September.
From the day of arrival at the experimental units until the end of the experiment, a strict daily routine was followed to allow optimal habituation of the fish (Fig. 4). All handlings (measurement of water quality parameters, renewal of the water, monitor mortality and morbidity) were limited to 30 min a day and were made predictable for the fish by a light cue (i.e. at 9:25 h, the light above every tank was lit, while between 9:30 h and 10 h, handlings were performed). Just prior to feeding, a camera was placed in the tanks to record feeding behavior. At 10 h, the fish were fed with commercially available pellets (Efico Sigma 862, pellet size 4.5 mm, Biomar SAS, France). The feeding response was recorded up to 1 h after feeding. Between 15 and 16 h the lights were lit again as mortality and morbidity were monitored.
The experimental design and housing was approved by the Ethical Committee of the Faculty of Veterinary Medicine and Bio-engineering Sciences, Ghent University (EC2017_92).
Experimental design
A schematic representation of the experimental design is provided in Fig. 4. Eight weeks (T-56) before the start of the experiment, all fish were anesthetized using Tricaine Methanosulfonate (MS-222; 100 mg L−1; Sigma Aldrich, Belgium). A T-bar anchor tag (Floy Tag Inc., USA) was inserted in the caudal epaxial musculature of all fish to allow individual identification. Hereby, all fish were photographed, weighed (WB), and measured (LB). After recovery, fish were replaced in their respective tanks where they could further habituate to the housing conditions and daily handling as described before.
At the start of the experiment (T0), three tanks were randomly assigned to the cortisol group (CORT) and three were assigned to the control group (CONT). The fish of the CORT group were fed with cortisol spiked feed (hydrocortisone, Sigma-Aldrich, USA) for the entire duration of the experiment (from T0 to T90). The spiked feed was prepared by uniformly spraying the pellets with cortisol dissolved in ethanol (1 mg mL−1) with a final concentration of 500 mg cortisol per kg feed, based on Aerts et al. [1]. Pellets were left to dry overnight at room temperature [1]. Fish of the CONT group were given the same amount ethanol-spiked feed without the addition of dissolved cortisol.
Sample collection
After 30 (T30) and 90 (T90) days, three fish per tank (i.e. nine fish per group) were euthanized using a stock solution of benzocaine (100 g benzocaine in 1 L ethanol) with a final concentration of 200 mg L−1 of seawater after which sampling was performed.
Blood was collected from each fish through caudal vessel lateral puncture using a heparinized needle and syringe. Time between catch and blood collection was on average 10 ± 4 min. Subsequently, all fish were measured (LE), weighed (WE) and pictures of pigmented and non-pigmented sides were collected. Fish were clinically inspected for the presence of skin lesions and/or deformities. Sagittal otoliths were obtained to determine the age of the fish (ICES, 2016). Wet mount preparations of gill biopsies and skin mucus samples were collected for parasitological examination. Scales from both the pigmented and non-pigmented side were collected and immediately frozen (- 20 °C) until cortisol analysis (see further).
Finally, a necropsy was performed for gross examination of internal organs. For histological examinations, samples of the gill, skin, liver, spleen, kidney, heart and intestines were collected. Formalin-fixed tissues were processed according to standard techniques, sectioned (5 µm) and stained with haematoxylin and eosin (H & E) or Periodic Acid Shiff (PAS).
Plasma and scale cortisol analysis
Heparinized tubes filled with blood were centrifuged (10 000 × g for 5 min at 5 °C). Plasma was collected and plasma cortisol concentration (PCC) was quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), derived from [1].
Scale cortisol concentration was quantified as described by Aerts and colleagues [1]. Briefly, after defrosting, mucus was removed, the scales were cut in smaller pieces and further homogenized using PowerBead tubes (ceramic 2.8 mm, Qiagen) and a bead ruptor (PowerLyzer 24, Qiagen) after which cortisol was extracted with methanol (VWR international BVBA, Belgium). After vortex-mixing and centrifugation (10 min, 3500 × g), the supernatant was collected and evaporated under nitrogen at 60 °C using a Turbovap nitrogen evaporator (Biotage, Sweden). The dried pellet was re-suspended and purified using a Gracepure™ C18 solid-phase extraction column after which UPLC-MS/MS (Xevo TQS, Waters, Millford, USA) analysis was performed.
Effect on fish health: secondary and tertiary responses correlated with increased cortisol concentration
Cellular and tissue response
Based on the histological samples of liver, gills and skin, cell and tissue related parameters were studied. As a proxy for metabolic effects, the degree of intrahepatocellular vacuolization in the liver was quantified based on H & E staining. An additional PAS staining was performed staining glycogen present in the vacuoles [35]. Five images of the stained histological sections were randomly acquired. The uncolored (H & E staining – lipid and/or glycogen storage) or purple (PAS staining – glycogen storage) area was calculated on each image using scientific image analysis software Image J (version 1.4). Based on a PAS staining of the gills, the number of goblet cells (GC) in gill filaments and lamellae were counted in five random gill filaments per fish. The results were expressed as the number of GC per 100 µm. The thickness of the epidermis was measured at 25 random chosen places based on H & E stained tissue samples using Image J software. To avoid bias during the assessment, image origins were blinded from the observers.
Hematology
Blood samples were used to estimate the osmotic balance and changes in hematological parameters. Plasma osmolality was determined using an osmometer (Osmomat 030, Gonotec, Berlin, Germany). For measuring hematocrit, the blood was dispensed in heparin containing micro-hematocrit tubes (GMBH + CO KG, Wertheim, Germany) and were centrifuged (10,000 g; 5 °C; 5 min). A blood smear was prepared using approximately seven µL of blood quickly after sampling. The smear was dried and stained using Hemacolor rapid staining (VWR, Belgium). The ratio between the total white blood cell (WBC) count and the total amount of red blood cells (RBC) was determined as a proxy for the immunological state. To estimate the WBC and RBC counts, blood smears were analyzed under the microscope and five pictures were collected per smear. Per picture, the number of WBC per 100 RBC was counted in three random fields. To avoid bias, the image origins were blinded from the observer.
Effects on organism level
Effects on organism level were evaluated using growth rate, body condition, feeding response (i.e., the total number of pellets eaten) and general health of the fish. The growth per day was calculated as the LE minus LB divided by the number of days the fish was in the experiment (30 or 90 days). The daily weight gain was calculated in a similar way (WE-WB). The Fulton condition index (K = 100*(W/L3) [12] was calculated at T-56 (KB) and at sampling (T30 or T90) (KE). Individual feeding response, measured as the number of pellets eaten per fish, was quantified using the video footage collected during feeding (10 h – 11 h). The general health of the fish was assessed by determining the presence of parasites based on the gill biopsies and skin mucus samples. In addition, gross and histological lesions in the internal organs were examined.
Statistical analysis
Statistical analysis of field measurements
Normality was evaluated using a QQ plot and, when needed, a logarithmic transformation of the data was performed to obtain normality. The difference between SCC in males and females was tested using a Welch two sample t-test. The correlation between the length, condition, weight, age, weight per scale, and SCC was studied using a Pearson’s product-moment correlation. The difference in SCC in fish with no lesions, skeletal deformities or skin ulcerations was tested using an ANOVA. Differences were considered to be significant when p-values were lower than 0.05. A p-value between 0.05 and 0.1 was considered a trend.
Statistical analysis of laboratory in vivo study
Normality was evaluated based on graphical evaluation (QQ plot) and, when needed, a logarithmic transformation of the data was performed to obtain normality. To ensure valid comparison between CONT and CORT fish, differences in fish-related characteristics (length, weight, condition, age) were analyzed using a linear mixed model (proc GLIMMIX) with tank as a random intercept. No differences were observed (Additional file 1) therefore allowing comparison between both groups without taking into account differences in fish-related characteristics.
The correlation between SCC and PCC was analyzed using a linear mixed model (proc GLIMMIX). All analyses were stratified by sampling day (T30/T90) and by group (CONT/CORT). The average SCC per fish was used calculated as the average of SCC of pigmented and non-pigmented side, unless specified otherwise.
Correlations between PCC, SCC and specified cellular, tissue, osmotic, hematological and whole-organism parameters were studied using a linear mixed model (proc GLIMMIX). SCC and PCC were used as response variables while other parameters were used as variables. All analyses were stratified by sampling day (T30/T90) and by group (CONT/CORT). The presence of parasites in skin or gills or gross and/or histological lesions in gills and internal organs was analyzed in a descriptive manner.
In all models, tank replicate was included as a random intercept. Differences were considered to be significant when p-values were lower than 0.05. A p-value between 0.05 and 0.1 was considered a trend. However, due to the low amount of fish sampled per group per sampling point (n = 9), p values below 0.1 might be suggestive of a significant difference although drawing clear conclusion would need more evidence. All statistical analyses were performed using SAS 9.4, graphical representations were constructed in R Studio.