Effects of Growth Hormone and Insulin-like Growth Factor I on Salinity Tolerance and Gill Na+, K+-ATPase in Atlantic Salmon (Salmo salar): Interaction with Cortisol (2023)

Triploidy is induced in Atlantic salmon (Salmo salar) to produce sterile fish for genetic containment and to hinder early sexual maturation in farmed fish, but it can have unwanted negative effects on growth, health, and welfare. However, the growth and welfare of triploid fish may be improved by adjusting the rearing environment, feeding conditions and diets. This study evaluated physiological changes and used a suite of biomarkers to assess the potential impact of diet on growth and welfare of diploid and triploid salmon during the parr-smolt transformation. Diploids and triploids, held at low temperature, were fed a standard salmon feed or one with hydrolyzed fish proteins thought to be suitable for triploid Atlantic salmon. Fish muscle was collected monthly from October to December (2454–3044 degree-days post-start feeding, ddPSF) for analysis of biomarkers, and the progress of the parr-smolt transformation was monitored using a seawater challenge test. Real-Time PCR and radioimmunoassay were used to assess growth and stress response biomarkers (expression of genes of the GH-IGF axis and HSP70; cortisol concentrations), and oxidative stress biomarkers of lipids (MDA) and proteins (AOPP) were assayed. Changes in the biomarkers were related to sampling time rather than being associated with diet or ploidy, and the changes were compatible with the progression of the parr-smolt transformation. Growth and expressions of the biomarkers in triploid Atlantic salmon were similar to those of their diploid counterparts, and there was no evidence that the rearing conditions employed in the study resulted in stress responses being elicited. Overall, the physiological indicators and biomarkers employed in this study did not point to there being any dietary effects on performance and welfare of diploid and triploid salmon that were undergoing parr-smolt transformation.

The differential effects of two seawater (SW) transfer regimes on the hypoosmoregulation, hormonal response, feed efficiency, and growth performance of 50g juvenile steelhead trout (Oncorhynchus mykiss) were determined to better understand the chronic responses to two salinity increase regimes. The fish reared in freshwater (FW) were exposed to either FW (control) or SW (32 ppt) via two regimes. In the first regime, the fish were exposed to a daily increase in salinity of 11 ppt and transferred to 32 ppt on the 3rd day (3DSW), and in the second regime, the fish were exposed to 20 ppt on the 1st day, which was followed by a daily 2 ppt increase, and transferred to 32 ppt on the 7th day (7DSW). Subsequently, an 8-week growth trial was conducted. At the end of the trial, the body weight of the control fish was higher than that of the 3DSW and 7DSW while the specific growth rate of the control was higher than that of the 3DSW but not significantly different from that of the 7DSW. Similarly, the feed efficiencies of the control were not different from that of the 7DSW but were higher than that of the 3DSW. The results on the plasma Na⁺ and Cl levels, osmolality, and gill Na⁺/K⁺ ATPase activity indicated that steelhead in the 3DSW and 7DSW developed high hypoosmoregulatory ability at weeks 4 and 8. Kidney Na⁺/K⁺-ATPase activity increased in the 7DSW and 3DSW groups at week 4. Circulating growth hormone and insulin-like growth factor I levels in the 7DSW group were elevated at week 4, whereas the cortisol level was not changed. Our results suggest that the 7DSW but not the 3DSW alleviated the chronic effect of osmotic stress on the growth rate and feed efficiency of 50g steelhead trout following SW transfer.

Euryhaline fishes are capable of maintaining osmotic homeostasis in a wide range of environmental salinities. Several pleiotropic hormones, including prolactin, growth hormone, and thyroid hormones (THs) are mediators of salinity acclimation. It is unclear, however, the extent to which THs and the pituitary-thyroid axis promote the adaptive responses of key osmoregulatory organs to freshwater (FW) environments. In the current study, we characterized circulating thyroxine (T4) and 3-3’-5-triiodothyronine (T3) levels in parallel with the outer ring deiodination (ORD) activities of deiodinases (dios) and mRNA expression of dio1, dio2, and dio3 in gill during the acclimation of Mozambique tilapia (Oreochromis mossambicus) to FW. Tilapia transferred from seawater (SW) to FW exhibited reduced plasma T4 and T3 levels at 6h. These reductions coincided with an increase in branchial dio2-like activity and decreased branchial dio1 gene expression. To assess whether dios respond to osmotic conditions and/or systemic signals, gill filaments were exposed to osmolalities ranging from 280 to 450 mOsm/kg in an in vitro incubation system. Gene expression of branchial dio1, dio2, and dio3 was not directly affected by extracellular osmotic conditions. Lastly, we observed that dio1 and dio2 expression was stimulated by thyroid-stimulating hormone in hypophysectomized tilapia, suggesting that branchial TH metabolism is regulated by systemic signals. Our collective findings suggest that THs are involved in the FW acclimation of Mozambique tilapia through their interactions with branchial deiodinases that modulate their activities in a key osmoregulatory organ.

Cortisol is the final product of the hypothalamic-pituitary-interrenal (HPI) axis and acts as a gluco- and mineralo-corticoid in fish. Long-term elevations of cortisol have been linked to reduced growth in fishes, but the mechanism(s) and relative sensitivities of species are still unclear. We carried out experiments to examine the relative effects of cortisol on growth and gill NKA activity in two salmonids: Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis). Treatment with intraperitoneal cortisol implants for 30days resulted in reduced growth in both species, but with greater sensitivity to cortisol in brook trout. Gill NKA activity was strongly upregulated by cortisol in Atlantic salmon, and weakly upregulated in brook trout but with no statistically significant effect. Cortisol treatment resulted in reduced plasma levels of insulin-like growth factor I and increased plasma growth hormone levels in Atlantic salmon. Our results demonstrate that there are species differences in the sensitivity of growth and osmoregulation to cortisol, even among species in the same family (Salmonidae).

Smoltification is a crucial step in the aquaculture of Atlantic salmon. Currently, the Na+/K+ ATPase (NKA) test is used to determine whether fish have adapted to seawater when moved from freshwater. The mortality rate of unadapted fish in Chile is ~10% after smoltification. During this period, BK channel expression decreases in gills after two weeks in saline water under laboratory conditions. However, these findings have not yet been tested on an industrial scale. We characterized the changes in mRNA levels of BK channels in gills from Salmo salar during smoltification using salmon farming protocols and compare these results against gill NKA activity and found a significant decrease in expression of BK channel transcripts during the smolt stage. We also characterized the dynamic of the BK channel expression (mRNA) versus NKA activity by using transcriptomic and RT-PCR analyses under an industrial setting. BK channels are involved in a variety of physiological processes, and our results indicate that BK potassium channel expression in gills could form the basis of a new complementary test to determine the level of smoltification more precisely.

Aquacultural Engineering, Volume 78, Part A, 2017, pp. 42-49

Fish live in close contact with microbes in their surrounding water. For cultivated marine fish larvae, stable microbial environments, characterized by slow-growing, K-selected bacteria, have been found to improve growth and survival. Compared to flow-through systems, recirculating aquaculture systems (RAS) offer a better possibility for obtaining microbial stability in the rearing water. However, the microbial ecology in RAS is complex and little studied, and to optimize the microbial water quality more knowledge is needed on microbial community dynamics and interactions between microbes and fish. In the present study we investigated microbial community dynamics in three large-scale RAS, operated with different salinities (12, 22 and 32ppt), for production of Atlantic salmon post-smolts. We focused on the stability of microbial communities over time in the bioreactors and the fish tanks, and compared the microbial communities within and between the three RAS. The study showed that: 1) The microbial community of the water was similar between the compartments of each RAS at the same time point, 2) The microbial communities of the water (and to a lesser extent the biofilm) within each system underwent large changes over time, 3) The microbiota of biofilm and water differed significantly within each system, 4) Biofilm community profiles were more similar between RAS than the water community profiles, and 5) Salinity structured the microbial community composition. The present study provides new knowledge on how the microbiota in RAS varies with time and how it is influenced by environmental factors.

General and Comparative Endocrinology, Volume 202, 2014, pp. 50-58

The present study assessed the influence of salinity and temperature on body growth and on muscle cellularity of Lophiosilurus alexaxdri vitelinic larvae. Slightly salted environments negatively influenced body growth of freshwater fish larvae and we observed that those conditions notably act as an environmental influencer on muscle growth and on local expression of hypertrophia and hypeplasia markers (IGFs and PCNA). Furthermore, we could see that salinity tolerance for NaCl 4gl⁻¹ diminishes with increasing temperature, evidenced by variation in body and muscle growth, and by irregular morphology of the lateral skeletal muscle of larvae. We saw that an increase of both PCNA and autocrine IGF-II are correlated to an increase in fibre numbers and fibre diameter as the temperature increases and salinity diminishes. On the other hand, autocrine IGF-I follows the opposite way to the other biological parameters assessed, increasing as salinity increases and temperature diminishes, showing that this protein did not participate in muscle cellularity, but participating in molecular/cellular repair. Therefore, slightly salted environments may provide adverse conditions that cause some obstacles to somatic growth of this species, suggesting some osmotic expenditure with a salinity increment.

Aquaculture, Volume 473, 2017, pp. 1-12

Aquaculture, Volume 463, 2016, pp. 1-6

Hippocampus reidi is one of the most popular seahorse species in the aquarium trade. The commercial breeding of this species is an alternative to reducing the fishing pressure on natural populations. Two experiments with newly born juveniles were conducted in this study to assess salinity tolerance (Lethal Time for 50% of the population-LT₅₀), survival and growth during the first 10days of life. A third experiment determined the isosmotic point of adults. The highest LT₅₀ was observed at 10psu, followed by 15, 20, 25, 30, and 35psu, and the lowest LT₅₀ was recorded at 5psu. A negative relationship between salinity and final weight and between salinity and final height was observed through the regressions performed, such that a decrease in salinity (until the lowest level analysed; 10psu) implied a higher growth in height and weight. The highest survival rates were observed between the salinities 10 and 25psu. The isosmotic point of the species was determined at 11.68psu (303.38mOsm/kg), which is in agreement with the salinity where the best salinity results were observed in the first two experiments. Using an intermediate salinity to produce H. reidi on a commercial scale would be advantageous because of survival and growth improvement in addition to requiring a lower seawater uptake and less cost for purchase artificial salt.

General and Comparative Endocrinology, Volume 229, 2016, pp. 84-91

Leptin is an anorexigenic peptide hormone that circulates as an indicator of adiposity in mammals, and functions to maintain energy homeostasis by balancing feeding and energy expenditure. In fish, leptin tends to be predominantly expressed in the liver, another important energy storing tissue, rather than in fat depots as it is in mammals. The liver also produces the majority of circulating insulin-like growth factors (IGFs), which comprise the mitogenic component of the growth hormone (GH)-IGF endocrine growth axis. Based on similar regulatory patterns of leptin and IGFs that we have documented in previous studies on hybrid striped bass (HSB: Morone saxatilis×Morone chrysops), and considering the co-localization of these peptides in the liver, we hypothesized that leptin might regulate the endocrine growth axis in a manner that helps coordinate somatic growth with energy availability. Using a HSB hepatocyte culture system to simulate autocrine or paracrine exposure that might occur within the liver, this study examines the potential for leptin to modulate metabolism and growth through regulation of IGF gene expression directly, or indirectly through the regulation of GH receptors (GHR), which mediate GH-induced IGF expression. First, we verified that GH (50nM) has a classical stimulatory effect on IGF-1 and additionally show it stimulates IGF-2 transcription in hepatocytes. Leptin (5 and/or 50nM) directly stimulated in vitro GHR2 gene expression within 8h of exposure, and both GHR1 and GHR2 as well as IGF-1 and IGF-2 gene expression after 24h. Cells were then co-incubated with submaximal concentrations of leptin and GH (25nM each) to test if they had a synergistic effect on IGF gene expression, possibly through increased GH sensitivity following GHR upregulation by leptin. In combination, however, the treatments only had an additive effect on stimulating IGF-1 mRNA despite their capacity to increase GHR mRNA abundance. This suggests that leptin’s stimulatory effect on GHRs may be limited to enhancing transcription or mRNA stability rather than inducing full translation of functional receptors, at least within a 24-h time frame. Finally, leptin was injected IP (100ng/g and 1μg/gBW) to test the in vivo regulation of hepatic IGF-1 and GHR1 gene expression. The 100ng/g BW leptin dose significantly upregulated in vivo IGF-1 mRNA levels relative to controls after 24h of fasting, but neither dosage was effective at regulating GHR1 gene expression. These studies suggest that stimulation of growth axis component transcripts by leptin may be an important mechanism for coordinating somatic growth with nutritional state in these and perhaps other fish or vertebrates, and represent the first evidence of leptin regulating GHRs in vertebrates.

Aquaculture, Volume 536, 2021, Article 736384

Preservation of amino acids is highly important, as these are vital nutrients that support many physiological and biochemical processes. During fish osmoregulation, the kidney reabsorbs amino acids and short peptides that are filtered from the blood into the nephron. However, little is known about reabsorption systems in teleost kidney. To identify transporters involved in this process, we examined by PCR the expression of eight genes, coding to peptide and amino acid transporters, as well as of two ion transport systems, in the kidney of the euryhaline Mozambique tilapia (Oreochromis mossambicus). We found that three amino acid transporters (slc1a5, ASCT2; slc7a9, b^0,+AT; slc6a19, B⁰AT) and one peptide transporter (slc15a2, PepT2) were expressed in the kidney, as were the two ion transporters VHA and NHE3 (coded by atp6v1a and slc9a3, respectively). Next, to investigate transcriptional interactions of these transporters with osmoregulation and nutritional status, we compared by qPCR expression levels in fish acclimated to either fresh or saltwater and at three time points after feeding (6, 24, 72h). Significant effect of salinity was detected on the expression of ASCT2 and VHA, whereas time after feeding affected PepT2 and VHA transcripts. Interactions between salinity and feeding significantly affected NHE3 transcription. PepT2 expression was an order of magnitude higher than free amino acid transporters, indicating the importance of peptide reabsorption in the kidney of Mozambique tilapia. Immunofluorescence staining of PepT2 showed an apical staining while, b^0,+AT antibody stained the basolateral membrane of the nephrons epithelial cells. The two transporters expression were found in separates segment of the nephrons. These results reveal specific transporters involved in kidney reabsorption of amino acids and short peptides and their response to the fish osmotic and nutritional status.