Pheromones are chemical substances that are secreted outwardly by organisms into their surroundings and received by the same species to influence organisms' behavioral habits, growth, development and so on activities. Scott et al. found that spermine in the semen of male sea lamprey(Petromyzon marinus) acts as a sex pheromone and attracts ovulating females(Scott et al., 2019). Zhu et al. showed that Large Yellow Croaker (Larimichthys crocea) are attracted to gut contents from conspecifics(Zhu et al., 2023). Pheromones can be involved in fish reproduction, migration, alarm and other behaviors that are important to the life of aquatic organisms(Kamio and Derby, 2017). Hexadecanedioic acid, xanthine, phenethylamine, proline and styrene in the aquatic metabolic group in this study were significantly different in MDvsLD, HDvsMD, HDvsLD, and the levels increased significantly with increasing density. Among them, hexadecanedioic acid and styrene are insoluble in water, and xanthine, phenethylamine and proline are soluble in water. Solubility determines the spatial extent of the pheromone, and since substances dissolved in water are more likely to diffuse in an aquatic environment, proline, xanthine, and phenethylamine are more likely to act as pheromones. Amino acids play a role in chemical communication as one of the few identified fish pheromones. Yambe et al. identified l-Kynurenine as a sex pheromone in the urine of ovulated female masu salmon(Oncorhynchus masou)(Yambe et al., 2006). Shoji et al. study finds that amino acids in stream water are essential for salmon (Oncorhynchus keta)homing migration(Shoji et al., 2003). However, it is important to note that the proline screened in this study was D-proline, which has been temporarily excluded as a natural pheromone. On the other hand, xanthine, as a typical purine and an important biomolecule, plays a crucial role in purine catabolic reactions(Liu et al., 2023). Xanthine can be converted to uric acid by the action of xanthine oxidase, and high levels of uric acid are associated with gout(Wu et al., 2021; Zhong et al., 2022). Therefore, the level of xanthine mainly indicates the health status of an organism and its levels in serum or urine can provide valuable information for the diagnosis and medical treatment of certain metabolic disorders(Pundir and Devi, 2014). Phenethylamine is an endogenous amine compound that can play an important biological role in the nervous system as a chemical messenger(Boulton, 1980; Branchek and Blackburn, 2003; Premont et al., 2001). Low concentrations of phenethylamine produce euphoria, but high concentrations of phenethylamine may form neurotoxic compounds(Edwards and Blau, 1973). Plasma phenethylamine concentrations are associated with stress, but they are quickly metabolized by monoamine oxygenase (MAO)(Grimsby et al., 1997; Paulos and Tessel, 1982). Studies have reported that the half-life of phenethylamine is very short in dogs (1.8-3 min) and is rapidly distributed and eliminated in rats after intravenous administration(Cone et al., 1978; Wu and Boulton, 1975). In the absence of any treatment, phenethylamine levels in organisms' tissues are very low (Durden and Boulton, 1982).
In the external environment, phenethylamine can also act as chemical cues to regulate individual animal behavior. Ferrero et al found that phenethylamine induced strong avoidance responses in rodent and herbivore species(Ferrero et al., 2011). Imre et al. showed that sea lamprey (Petromyzon marinusalso) showed a strong avoidance response to phenethylamine(Imre et al., 2014). Bredy and Barad showed that phenethylamine can act as a pheromone to communicate information about fear or threats(Bredy and Barad, 2009). Phenethylamine normally binds to the vertebrate trace amine-associated receptor (TAAR), and its perception by the TAAR4 olfactory receptor leads to an increase in intracellular cAMP levels. cAMP directly activates cyclic nucleotide-gated channels (CNG channels) to allow the entry of Na+ and Ca2+, depolarizing olfactory sensory neurons (OSN) to generate action potentials and converting chemical signals into electrical signals(Mombaerts et al., 1996; Xu and Li, 2020).The electrical signal is transmitted to brain regions to produce olfactory perception, thus enabling the transmission of information(Lindemann and Hoener, 2005). Phenethylamine levels have been shown to increase in the urine of stressed animals(Paulos and Tessel, 1982; Snoddy et al., 1985). In this study, the levels of phenethylamine were found to increase with increasing density. Subsequently, we examined changes in key genes of the HPI axis and GH/IGF-1 signaling pathway as well as physiological indicators in turbot after density treatment and phenethylamine treatment and performed correlation analysis.
The HPI axis plays an important role in the response of fish to environmental stresses(Rotllant et al., 2000). In a stressful state, the HPI axis is first activated, causing the body to release large amounts of cortisol in response to the stressor(Barton, 2002; Yusishen et al., 2020). Excessive cortisol will induce secondary and tertiary stress responses, resulting in physiological and other functional disorders in fish (Van Der Boon et al., 1991). Bi et al. found that the serum ACTH and cortisol levels of hybrid sturgeon (♀Acipenser baerii×♂Acipenser schrenckii)increased with increasing stocking density (Bi et al., 2023). Wang et al. found that a stocking density of 24 kg/m3 for 220 days resulted in a significant increase in plasma cortisol levels in Atlantic salmon when compared to a density of 6 kg/m3(Wang et al., 2019). In addition, changes in other environmental factors, such as ammonia exposure, nitrate exposure, and pathogenic infections, can also upregulate CRH, ACTH genes, and plasma cortisol in fish(Jia et al., 2017; Madison et al., 2013; Yu et al., 2021). This study also showed that plasma cortisol was significantly higher in the HD group compared to the MD and LD groups, while the expression levels of CRH and ACTH genes also increased with increasing density. Similarly, plasma cortisol was significantly higher in the HP group compared to the LP and CON groups, while the expression levels of CRH and ACTH genes also increased with increasing phenethylamine concentrations. The similarity between the phenethylamine treatment and density treatment suggests that high stocking density may activate the turbot HPI axis through phenethylamine, leading to a stress response. Under stress, fish release large amounts of cortisol, which increases the metabolism of carbohydrates, fats and proteins and controls the flow of energy in the organism in response to the stressor (Barton, 2002; Flik et al., 2006; Mommsen et al., 1999; Shepherd et al., 2018). Plasma glucose, lactate, and triglycerides were significantly elevated in both HD and HP groups in this study, which may be due to energy mobilization by the organism to resist the unfavorable external environment, corroborating with the above results.
In teleost fish, the GH/IGF-1 signaling pathway regulates a variety of physiological functions, such as growth, reproduction, immunity, and osmoregulation(Blanco, 2020; Canosa and Bertucci, 2023; Pérez-Sánchez et al., 2018). GH levels in fish are positively associated with growth in vivo. However, under prolonged stress conditions, GH levels in fish can fluctuate, disrupting the function of the GH/IGF-1 signaling pathway. Environmental factors such as temperature, salinity, density, and other breeding-induced discomfort can cause a decrease in fish IGF-1 levels, often resulting in an impact on fish growth and development (Davis and Peterson, 2006; Deane et al., 2002; Seo and Park, 2022). The present study found that both GH and IGF-1 were significantly downregulated in the HD and HP groups, suggesting the inhibitory effects of high density and phenethylamine treatment on turbot growth. Thyroid hormones (TH) have also been shown to play a crucial regulatory role in fish growth, often working synergistically with other hormones(Power et al., 2001; Xie et al., 2015). In fish, TH exerts its biological function mainly through the formation of T3(Yamano, 2005). Therefore, we evaluated the T3 levels in turbot plasma. The results showed that T3 increased with the increase of density. Ardiansyah and Fotedar found that the T3 of juvenile barramundi (Lates calcarifer Bloch) decreased gradually with the increase of culture density(Ardiansyah and Fotedar, 2016). This may be due to the short duration of density treatment (15 days), where the fish are at an early level of stress and T3 is elevated to promote energy metabolism in response to the unfavorable environment. T3 also increased with increasing phenethylamine concentration in the phenethylamine treatment. The effects of density treatment and phenethylamine treatment on turbot growth were also highly consistent. Interestingly, in our study, GH gene expression was almost absent in both the HD and HP groups, but IGF-1 gene expression was still present. This may be because the HD and HP groups promoted IGF-1 expression by elevated T3 acting on the liver. It has been shown that T3 increases IGF-1 mRNA expression and stimulates the release of IGF-1.(Pepene et al., 2001; Robson et al., 2002).
Phenethylamine concentration was strongly correlated with stocking density, and the effects of phenethylamine treatment and density treatment on the HPI axis, GH/IGF-1 signaling pathway, and key physiological indicators (cortisol, T3, glucose, triglycerides) were highly similar. In the present study, phenethylamine was detected at LD, MD and HD, but only the turbot in the HD group produced significant stress and growth inhibition. This suggests that the effects of phenethylamine (harmful or beneficial) are dose dependent under specific conditions. In the phenethylamine treatment experiment, even the LP group (10− 7 mol/l) had a significant negative effect on turbot, which indicates that phenethylamine has a trace effect.