Aquaculture is an important component of a complex global food system, through which 52% of the fish are supplied for human consumption [1]. Fish farming and production essentially depends on reproduction, with the generation of monosex populations being one of the primary breeding technologies [2]. As significant dimorphism commonly exists between fish sexes in terms of growth, size, color, and other economically relevant characteristics [3,4]. Many countries/companies endeavor to create monosex (all-male or all-female) aquaculture populations, or produce high ratios of males or females that have higher value [2,5,6]. It is well known that gonadal sex differentiation in teleost fish exhibits a high degree of plasticity contrast to other vertebrates, as it can be influenced by a variety of environmental factors such as temperature, pH, or photoperiod, etc. [7,8]. The influences of these environmental factors on sex differentiation in fish have been thoroughly investigated [[9], [10], [11]]. Consequently, changing sexual differentiation is a convenient and feasible method to improve the economic value of aquaculture species.

NF-κB is a key transcription factor that is involved in numerous cellular responses to a wide range of signals. As an important molecule for cell survival and death in the immune system, it also controls the processes involved in reproduction and sexual differentiation [12,13]. It has been confirmed that the activation of NF-κB can down-regulate the expression of Mullerian inhibiting substance (MIS) in mammals [13]. The MIS is also known as an anti-Mullerian hormone (AMH), which is essential in normal sex differentiation and reproductive functions [14]. In mammals, NF-κB is directly involved in controlling the proliferation and secretory activities of porcine ovary cells [15] and is implicated in the control of follicular development in rodents [16]. In fish, NF-κB has also been proved to be involved in the gonad differentiation. NF-κB has been shown to inhibit cell apoptosis during gonadal transformation; thus, promoting female bias in zebrafish [17]. Further, NF-κB plays a critical role in the gonadal development of Lates calcarifer through small-scale transcriptomics [18]. Currently, it has been hypothesized that QNZ affects calcium entry through store-operated channels (SOCs) [19], which are known to be required for the initiation of the NF-κB signaling pathway [[20], [21], [22]]. As a quinazoline derivative, it is more effective in blocking the NF-κB signaling pathway than others [23]. Therefore, investigation of the NF-κB signaling pathway with QNZ can be utilized as a practical breakthrough point to control sex differentiation in fish.

Paramisgurnus dabryanus (Cypriniformes: Cobitidae) (namely the large-scale loach) is a typically sexual dimorphism fish [24]. To date, it has become an important commercial cultured animal in China [25]. Simultaneously, the dissimilar gonad differentiation times between male and female individuals, as well as the complex sex determination mechanisms of large-scale loaches, make them excellent models for the study of sex differentiation in fish. Consequently, our laboratory conducted gonadal transcriptomic analysis on adult large-scale loaches with QNZ immersion exposure in preliminary, tests and found that several genes correlated with gametogenesis, fertilization, and gonadal differentiation changed significantly following QNZ treatment. This suggested that the QNZ treatment could impact individual reproduction and gonadal differentiation to a certain extent. Based on existing laboratory research, this study initially conducted a long-term immersion exposure with juvenile large-scale loaches during the critical period of gonadal differentiation (14–150 day post fertilization (dpf)) to analyze changes in the gonadal tissues and sex ratios, the same were employed, exposure concentrations were increased, whereafter the gonadal tissue, sex hormone levels, sperm activities and offspring development were analyzed to further study the effects of QNZ on the gonadal differentiation and reproductive capacities of adult large-scale loaches. To the best of our knowledge, this study is the first to comprehensively investigate the impacts of the inhibition of the NF-κB signaling pathway on the gonadal differentiation and sex ratio of fish, which more deeply elucidates the control of fish sexes in aquaculture.