1. Introduction
CO2 regulation is central in aquaculture, and particularly high-density aquaculture, where fish metabolic activities may elevate CO­2 (Hillet al . 2004; Summerfelt et al . 2000) to levels 10–40 times greater than in the ocean (Fivelstad et al . 2003; Fosset al . 2003; Steffensen & Lomholt 1988). Elevated CO2 can significantly affect fish health and growth (Fivelstad et al . 2003; Foss et al . 2003; Steffensen & Lomholt 1988). Although recirculating aquaculture systems provide efficient environmental control and visibility, allowing optimal production efficiency (Ebeling & Timmons 2012), farmed fish are more susceptible than wild fish to external stressors (Kvamme et al . 2013).
In fish, exposure to elevated CO2 is counteracted by increased respiration amplitude and frequency (Gilmour and Perry 2006). This can, in turn, cause chronic excess O2 levels, thus indirectly reducing growth. Prolonged exposure of farmed fish to elevated CO2 leads to hypercarbia and respiratory acidosis, reducing feed intake and growth (Fivelstad et al . 2007) and fertility (Ben-Asher et al . 2013), and causing renal calcium deposition (Fivelstad et al . 2018). In marine fish, elevated CO2 causes metabolic acidosis (Bernier and Randall 1998), ionic imbalance (Brauner et al . 2000), stress hormone activation (Iwama et al . 1989), respiratory acidosis, and excess reactive oxygen species (ROS) production, leading to oxidative cellular damage (Cao et al . 2010). To prevent oxidative stress, the antioxidant enzyme system must be activated (Song et al . 2017). CO2 can affect liver biosynthesis (Rognstad 1983; Stappet al . 2015), increasing the incidence of lymphocytic portal hepatitis (Good et al . 2010) and liver tissue damage (Frommelet al . 2012).
Exposure to elevated CO2 affects fish growth, and tolerance to CO2 varies among species (Martens et al . 2006). Salmo salar (Atlantic salmon) growth decreases linearly with increasing CO2 (Fivelstad et al . 2018; Khan et al . 2018; Mota et al . 2019). In Atlantic cod, exposure to 18 mg/L CO2 affected growth and cataract incidence (Moran and Støttrup 2011; Neves and Brown 2015).Scophthalmus maximus growth declined by 26% on exposure to 21 mg/L CO2 (Stiller et al . 2015).
The pituitary and thyroid glands secrete growth hormone (GH) and thyroxine, which act downstream of the liver, constituting the hypothalamic–pituitary–thyroid–liver axis. The liver is the primary metabolic organ, with key roles in metabolic homeostasis, metabolism, endocrine regulation, endogenous compound degradation, and detoxification, among other functions (Almroth et al . 2019). Chronic stress can lead to hepatic insufficiency. As lipids accumulate in hepatocytes, microscopic changes occur in the liver, eventually leading to macroscopic lesions (Bolla et al .​​ 2011). Liver tissue expresses large amounts of various proteins, and contains high levels of antioxidant enzymes and antioxidants. Liver enzymes can be used to assess liver function (Casas-Grajales & Muriel 2015; Kaplowitz 1981). The liver is a major source of endogenous type-1 insulin-like growth factor (IGF-I), which can be used to assess fish growth and health; IGF is associated with growth, metabolism, development, cell differentiation, reproduction, osmoregulation, and immune response in fish (Reinecke 2005). In the hypothalamic–pituitary–thyroid–liver axis, pituitary secretion of GH stimulates hepatic synthesis and the release of IGF-I, which specifically inhibits GH gene transcription and secretion via negative feedback (Wallenius 2001). Pituitary GH/hepatic IGF-I is an important endocrine regulatory axis (Reinecke 2005, 2006) and important signaling pathway in the thyroid–liver axis.
Thyroid hormones regulate GH expression in the pituitary gland, and thyroid hormone response elements are present in the GH gene promoter in mammals (Forhead 2000) and in scleractinian fish (Eppler 2011). In mammals, GH controls growth and development by mediating IGF-I secretion (Daughaday & Rotwein 1989; Eppler 2011; Schmid 2000). Thyroid hormone, GH, IGF-1, IGF-1 receptor (IFG-1R), and other hormones affect growth, development, differentiation, metabolism, immunity, and other processes (Ömer 2011). Although studies have addressed liver responses to toxicants such as ammonia (Cheng et al . 2015), zinc oxide (Horieet al . 2020), and nitrate (Yu et al . 2021), responses to CO2 have not previously been studied.
Scophthalmus maximus (turbot; Pleuronectiformes [flounders], Scophthalmidae), a key aquaculture species in China (particularly northern China), has high economic value and market demand, and is farmed mainly in recirculating aquaculture systems. Using this species, we applied a CO2 gradient to evaluate responses in terms of growth, health, and oxygen-carrying capacity. Further, we evaluated the role of endocrine function (in terms of GHR and IGF expression) in CO2-induced growth retardation. Elevated CO2 (32 mg/L) negatively affected juvenileScophthalmus maximus growth and health. Growth can be hampered even at CO2 concentrations below 8 mg/L. This study provides valuable insights into the growth of juvenileScophthalmus maximus under CO2 stress.