GH–IGF system regulation of attenuated muscle growth and lipolysis in Atlantic salmon reared at elevated sea temperatures

Growth regulation in adult Atlantic salmon (1.6 kg) was investigated during 45 days in seawater at 13, 15, 17, and 19 °C. We focused on feed intake, nutrient uptake, nutrient utilization, and endocrine regulation through growth hormone (GH), insulin-like growth factors (IGF), and IGF-binding proteins (IGFBP). During prolonged thermal exposure, salmon reduced feed intake and growth. Feed utilization was reduced at 19 °C after 45 days compared with fish at lower temperatures, and body lipid storage was depleted with increasing water temperature. Although plasma IGF-1 concentrations did not change, 32-Da and 43-kDa IGFBP increased in fish reared at ≤17 °C, and dropped in fish reared at 19 °C. Muscle igf1 mRNA levels were reduced at 15 and 45 days in fish reared at 15, 17, and 19 °C. Muscle igf2 mRNA levels did not change after 15 days in response to increasing temperature, but were reduced after 45 days. Although liver igf2 mRNA levels were reduced with increasing temperatures after 15 and 45 days, temperature had no effect on igf1 mRNA levels. The liver igfbp2b mRNA level, which corresponds to circulating 43-kDa IGFBP, exhibited similar responses after 45 days. IGFBP of 23 kDa was only detected in plasma in fish reared at 17 °C, and up-regulation of the corresponding igfbp1b gene indicated a time-dependent catabolic response, which was not observed in fish reared at 19 °C. However, higher muscle ghr mRNA levels were detected in fish at 17 and 19 °C than in fish at lower temperatures, indicating lipolytic regulation in muscle. These results show that the reduction of muscle growth in large salmon is mediated by decreased igf1 and igf2 mRNA levels in addition to GH-associated lipolytic action to cope with prolonged thermal exposure. Accordingly, 13 °C appears to be a more optimal temperature for the growth of adult Atlantic salmon at sea.     

Daily rhythms in the somatotropic axis of Senegalese sole (Solea senegalensis): The time of day influences the response to GH administration

Growth factors in vertebrates display daily rhythms, which, while widely described in mammals, are still poorly understood in teleost fish. Here, we investigated the existence of daily rhythms in the somatotropic axis of the flatfish Solea senegalensis. In a first experiment, daily rhythms of the expression of pituitary adenylate cyclase–activating polypeptide (pacap), growth hormone (gh), insulin-like growth factor 1 (igf1) and its receptor (igf1r) were analyzed under a 12:12 h light:dark cycle. All genes displayed daily rhythms with the acrophases of pacap, gh and igf1 located in the second half of the dark phase (ZT 20:28–0:04 h), whereas the acrophase of igf1r was located around mid-light (ZT 5:33 h). In a second experiment, the influence of the time of day (mid-light, ML, versus mid-darkness, MD) of GH administration on the expression of these factors and on plasma glucose levels was tested. The response observed depended on the time of injection: the strongest effects were observed at MD, when GH administration significantly reduced pituitary gh and enhanced liver igf1 expression. These results provide the first evidence of daily rhythms and differential day/night effects in growth factors in S. senegalensis, suggesting new insights for investigating the physiology of growth and possible applications to improve fish aquaculture.     

Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)

Eurasian perch (Perca fluviatilis) is a promising aquaculture candidate, but the growth performance of this non-domesticated species may be negatively affected by its stress responsiveness to intensive culture conditions. To evaluate this potential problem, juvenile Eurasian perch were exposed to a standardized handling stressor twice a week for an 8-week period. A similar study was conducted on domesticated rainbow trout (Oncorhynchus mykiss) for comparison of intra- and inter-specific differences. The stressed fish of both species showed lower body growth than the non-stressed control fish, however, the final mean body mass was 35.4% lower in the stressed Eurasian perch than in the non-stressed controls, compared to 22.8% difference between the two groups in rainbow trout. The stress responsiveness was examined by comparing the post-stress cortisol and glucose levels in repeatedly stressed fish and fish exposed to the stressor only once. The cortisol stress response in both species strongly indicated a habituation to the repeated stressor. Thus, repeatedly stressed Eurasian perch reached maximum cortisol levels of 130 ng/mL after 0.5 h compared to 200 ng/mL in the fish stressed once, while considerably smaller differences in cortisol levels were shown between the repeatedly and single stressed rainbow trout. Rainbow trout also showed lower post-stress glucose levels in the repeatedly stressed fish compared to the single stressed fish. In contrast, the glucose levels in both groups of Eurasian perch increased abruptly after stress treatment and remained elevated at approximately 19 mM for 6 h; levels were three times as high as the peak levels 3 h post-stress in rainbow trout. Together, the habituation of the stress response shown in both species did not eliminate the growth difference found in the repeatedly stressed fish versus the control fish. Further, the lower growth performance of Eurasian perch compared to rainbow trout could partly be due to the increased energy consumption in the more stress responsive Eurasian perch.     

Disease resistance and health parameters of growth-hormone transgenic and wild-type coho salmon,Oncorhynchus kisutch

To extend previous findings regarding fish health and disease susceptibility of growth-enhanced fish, hematological and immunological parameters have been compared between growth hormone (GH) transgenic and wild-type non-transgenic coho salmon (Oncorhynchus kisutch). Compared to non-transgenic coho salmon, transgenic fish had significantly higher hematocrit (Hct), hemoglobin (Hb), mean cellular hemoglobin (MCH), mean cellular volume (MCV), and erythrocyte numbers, and lower white cell numbers. In addition, resistance to the bacterial pathogen Aeromonas salmonicida (causal agent of furunculosis) has been assessed between the strains. Higher susceptibility of transgenic fish to this disease challenge was observed in two separate year classes of fish. The present findings provide fundamental knowledge of the disease resistance on GH enhanced transgenic coho salmon, which is of importance for assessing the fitness of transgenic strains for environmental risk assessments, and for improving our understanding effects of growth modification on basic immune functions.     

Regulation of feeding behavior and food intake by appetite-regulating peptides in wild-type and growth hormone-transgenic coho salmon

Survival, competition, growth and reproductive success in fishes are highly dependent on food intake, food availability and feeding behavior and are all influenced by a complex set of metabolic and neuroendocrine mechanisms. Overexpression of growth hormone (GH) in transgenic fish can result in greatly enhanced growth rates, feed conversion, feeding motivation and food intake. The objectives of this study were to compare seasonal feeding behavior of non-transgenic wild-type (NT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch), and to examine the effects of intraperitoneal injections of the appetite-regulating peptides cholecystokinin (CCK-8), bombesin (BBS), glucagon-like peptide-1 (GLP-1), and alpha-melanocyte-stimulating hormone (α-MSH) on feeding behavior. T salmon fed consistently across all seasons, whereas NT dramatically reduced their food intake in winter, indicating the seasonal regulation of appetite can be altered by overexpression of GH in T fish. Intraperitoneal injections of CCK-8 and BBS caused a significant and rapid decrease in food intake for both genotypes. Treatment with either GLP-1 or α-MSH resulted in a significant suppression of food intake for NT but had no effect in T coho salmon. The differential response of T and NT fish to α-MSH is consistent with the melanocortin-4 receptor system being a significant pathway by which GH acts to stimulate appetite. Taken together, these results suggest that chronically increased levels of GH alter feeding regulatory pathways to different extents for individual peptides, and that altered feeding behavior in transgenic coho salmon may arise, in part, from changes in sensitivity to peripheral appetite-regulating signals.     

Disease resistance, stress response and effects of triploidy in growth hormone transgenic coho salmon

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non‐transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non‐transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non‐transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth‐accelerated coho salmon.     

Changes in free and total plasma cortisol levels in juvenile haddock (Melanogrammus aeglefinus) exposed to long-term handling stress

We measured changes in free and total plasma cortisol levels, plasma glucose, gill hsp70 levels, and growth in haddock (Melanogrammus aeglefinus) subjected to a long-term handling stress (15 s out of water, each day, for 4 weeks), and the effect of this long-term stress on the ability of haddock to respond to an acute stressor. The acute stressor was a single handling stress, and fish were sampled at 1, 6, and 12 h post-stress. During the long-term stress study, free and total plasma cortisol levels increased significantly (10-fold) in the stressed group after the second week. However, the percentage of free cortisol was already significantly elevated by the first week (control 17%, stressed 55%), and remained high during the second week (control 35% and stressed 65%). After 3 and 4 weeks of handling, both free and total cortisol declined in stressed fish to levels that were not significantly different from pre-stress values. Control fish grew significantly more than stressed fish (by 32% and 18%, respectively) over the 4 week study, and condition factor only increased in control fish. Although fish from the control group showed elevated total plasma cortisol levels (to 47 ng mL(-1)) 1 h after the acute stress, and the levels in stressed fish were comparable to those for the control fish, no significant increase in plasma cortisol was measured in the group subjected to the long-term stress. Free plasma cortisol levels did not increase significantly in either group following the acute stress. However, free plasma cortisol levels were significantly higher in long-term stress group, as compared with the control group, at 6 h post-stress. Plasma glucose and gill hsp70 levels were not altered by either the long-term stress or acute stressor. Our data indicate that cortisol (free and total), but not glucose or hsp70, appears to be adequate to assess short- and long-term stress in haddock.     

Oxidative stress in growth hormone transgenic coho salmon with compressed lifespan – a model for addressing aging

Abstract

Growth hormone (GH) transgenic fish have dramatically enhanced growth rates, increased oxygen demands and reactive oxygen species production. GH-transgenic coho salmon provide an opportunity to address effects of increased metabolism on physiological aging. The objective of this study was to compare oxidative stress in wild-type (WT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch) of different ages (1 and 2 years). Antioxidant enzyme activity, protein carbonyls (PC) and glutathione (GSH, GSSG) were measured. PC correlated to growth rates in individual fish. T fish exhibited lower antioxidant enzyme activities and GSH levels compared to the WT, while levels of PC and GSSG were higher. Age affects were observed in both WT and T fish; enzyme activities and GSH decreased while PC and GSSG increased. Our results support the metabolic rate theory of aging. This study aims to be a platform for continued studies of the theories of aging using fish as model organisms.     

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.     

Oxidative stress in growth hormone transgenic coho salmon with compressed lifespan - a model for addressing aging

Abstract Growth hormone (GH) transgenic fish have dramatically enhanced growth rates, increased oxygen demands and reactive oxygen species production. GH-transgenic coho salmon provide an opportunity to address effects of increased metabolism on physiological aging. The objective of this study was to compare oxidative stress in wild-type (WT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch) of different ages (1 and 2 years). Antioxidant enzyme activity, protein carbonyls (PC) and glutathione (GSH, GSSG) were measured. PC correlated to growth rates in individual fish. T fish exhibited lower antioxidant enzyme activities and GSH levels compared to the WT, while levels of PC and GSSG were higher. Age affects were observed in both WT and T fish; enzyme activities and GSH decreased while PC and GSSG increased. Our results support the metabolic rate theory of aging. This study aims to be a platform for continued studies of the theories of aging using fish as model organisms.     

Effects of aquaculture related stressors and nutritional restriction on circulating growth factors (GH, IGF-I and IGF-II) in Atlantic salmon and rainbow trout

The effects of aquaculture related stressors on circulating levels of GH, IGF-I and for the first time, IGF-II in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) were investigated. Specifically, circulating growth factor levels were measured in four different experiments. Two 24 h confinement stressor procedures, (one with Atlantic salmon, the other with rainbow trout); following a hypo-osmotic stressor (freshwater bath) in salt water acclimated, adult, Atlantic salmon; and during a 22 day starvation and re-feeding protocol with juvenile Atlantic salmon. Handling and confinement resulted in significant decreases in circulating levels of all three growth factors in Atlantic salmon, and IGF-I and IGF-II (but not GH) in rainbow trout. A 2-3 h freshwater bath to remove gill parasites on a commercial Atlantic salmon aquaculture operation caused a significant decrease in circulating GH and IGF-I concentrations, but no significant change in IGF-II concentration, 2 days post bathing. Starvation for a period of 15 days in Atlantic salmon resulted in a significant increase in circulating GH levels and a significant decrease in circulating IGF-I and IGF-II. Re-feeding of starved fish for 7 days resulted in a significant decrease in GH to the concentration measured in continually fed fish, however re-feeding did not change plasma levels of IGF-I and IGF-II relative to continually starved fish. The results presented here confirm previously observed handling and confinement stressor induced effects on GH and IGF-I and, for the first time, on IGF-II in salmonids. Furthermore this study confirms the nutritional regulation of GH, IGF-I and IGF-II in juvenile Atlantic salmon.     

Growth hormone and seawater adaptation in coho salmon, Oncorhynchus kisutch

1. Effects of growth hormone (GH) were examined on short-term aspects of seawater adaptation in coho salmon smolts. 2. Injection of somatostatin (SRIF) immediately prior to seawater entry suppressed plasma GH levels, but did not have any significant effects at 6 or 12 hr on hematocrits, plasma glucose or plasma Na+ levels. 3. Plasma GH levels increased 250% within 36 hr after seawater exposure. 4. Plasma glucose levels, in contrast, were significantly lower in the seawater fish after 36 hr post-exposure. 5. Plasma Na+ levels increased to 190 mEq/1 by 24 hr but subsequently returned to freshwater levels while hematocrits showed no significant changes over the 72 hr of exposure. 6. The significance of these results is discussed in terms of successful seawater adaptation in coho salmon.     

Gill Morphometry in Growth Hormone Transgenic Pacific Coho Salmon,Onchorhynchus kisutch,Differs Markedly from that in GH Transgenic Atlantic Salmon

In a previous study we showed that many of the morphological features of the respiratory system of GH (growth hormone) transgenic Atlantic salmon are greater than similarly sized control salmon. Here we show that the manifestation of GH transgene is similar in two different lines of GH transgenic Pacific coho salmon, but that it is very different from that in the GH transgenic Atlantic salmon. The GH transgenic Pacific coho salmon do not have a larger gill surface area than similarly sized control fish.     

Upper thermal tolerance of wild‐type,domesticated and growth hormone‐transgenic coho salmon Oncorhynchus kisutch

In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26·9° C, C_Tmax) were observed among size‐matched wild‐type, domesticated, growth hormone (GH)‐transgenic fish fed to satiation, and GH‐transgenic fish on a ration‐restricted diet. Instead, GH‐transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17·3 ± 0·1° C, T_AB). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH‐transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits.     

Gene expression in the liver of rainbow trout, Oncorhynchus mykiss, during the stress response

To better appreciate the mechanisms underlying the physiology of the stress response, an oligonucleotide microarray and real-time RT-PCR (QRT-PCR) were used to study gene expression in the livers of rainbow trout (Oncorhynchus mykiss). For increased confidence in the discovery of candidate genes responding to stress, we conducted two separate experiments using fish from different year classes. In both experiments, fish exposed to a 3 h stressor were compared to control (unstressed) fish. In the second experiment some additional fish were exposed to only 0.5 h of stress and others were sampled 21 h after experiencing a 3 h stressor. This 21 h post-stress treatment was a means to study gene expression during recovery from stress. The genes we report as differentially expressed are those that responded similarly in both experiments, suggesting that they are robust indicators of stress. Those genes are a major histocompatibility complex class 1 molecule (MHC1), JunB, glucose 6-phosphatase (G6Pase), and nuclear protein 1 (Nupr1). Interestingly, Nupr1 gene expression was still elevated 21 h after stress, which indicates that recovery was incomplete at that time.     

Elevated CO2 impairs olfactory‐mediated neural and behavioral responses and gene expression in ocean‐phase coho salmon (Oncorhynchus kisutch)

Elevated concentrations of CO₂ in seawater can disrupt numerous sensory systems in marine fish. This is of particular concern for Pacific salmon because they rely on olfaction during all aspects of their life including during their homing migrations from the ocean back to their natal streams. We investigated the effects of elevated seawater CO₂ on coho salmon (Oncorhynchus kisutch) olfactory‐mediated behavior, neural signaling, and gene expression within the peripheral and central olfactory system. Ocean‐phase coho salmon were exposed to three levels of CO₂, ranging from those currently found in ambient marine water to projected future levels. Juvenile coho salmon exposed to elevated CO₂ levels for 2 weeks no longer avoided a skin extract odor that elicited avoidance responses in coho salmon maintained in ambient CO₂ seawater. Exposure to these elevated CO₂ levels did not alter odor signaling in the olfactory epithelium, but did induce significant changes in signaling within the olfactory bulb. RNA‐Seq analysis of olfactory tissues revealed extensive disruption in expression of genes involved in neuronal signaling within the olfactory bulb of salmon exposed to elevated CO₂, with lesser impacts on gene expression in the olfactory rosettes. The disruption in olfactory bulb gene pathways included genes associated with GABA signaling and maintenance of ion balance within bulbar neurons. Our results indicate that ocean‐phase coho salmon exposed to elevated CO₂ can experience significant behavioral impairments likely driven by alteration in higher‐order neural signal processing within the olfactory bulb. Our study demonstrates that anadromous fish such as salmon may share a sensitivity to rising CO₂ levels with obligate marine species suggesting a more wide‐scale ecological impact of ocean acidification.