Molecular Mechanisms of Tebuconazole Affecting the Social Behavior and Reproduction of Zebrafish

Abstract

The aim of this study was to explore the underlying mechanism of adverse effects caused by tebuconazole (TEB) on the reproduction of aquatic organisms In the present study, in order to explore the effects of TEB on reproduction, four-month-old zebrafish were exposed to TEB (0, DMSO, 0.4 mg/L, 0.8 mg/L, and 1.6 mg/L) for 21 days. After exposure, the accumulations of TEB in gonads were observed and the cumulative egg production was evidently decreased. The decline of fertilization rate in F1 embryos was also observed. Then the changes in sperm motility and histomorphology of gonads were discovered, evaluating that TEB had adverse effects on gonadal development. Additionally, we also found the alternations of social behavior, 17β-estradiol (E2) level, and testosterone (T) level. Furthermore, the expression levels of genes involved in the hypothalamic-pituitary-gonadal (HPG) axis and social behavior were remarkably altered. Taken together, it could be concluded that TEB affected the egg production and fertilization rate by interfering with gonadal development, sex hormone secretion, and social behavior, which were eventually attributed to the disruption of the expressions of genes associated with the HPG axis and social behavior. This study provides a new perspective to understanding the mechanism of TEB-induced reproductive toxicity.

Keywords: HPG axis; mechanism; reproductive toxicity; social behavior; tebuconazole; zebrafiah.

Figure 1

Cumulative egg production of zebrafish in 14 days before exposure (A) and 21 days (B) during exposure (3 pairs of fish per replicate, 3 replicates). (C) Fertilization rates of F1 embryos from the parents exposed to TEB (200 larvae per replicate, 4 replicates). Data are expressed as the mean ± SEM of three independent experiments. Asterisks in the figures were regarded as statistical differences between the exposure groups and control group (Tukey: * p < 0.05).

Figure 2

Effects of TEB on the gonadal development of zebrafish. (A) sections of ovary and testis stained by H&E (mean ± SEM, n = 6). Bars represent 50 µm (900×) and 500 µm (90×). Percentages of primary oocytes (PO), cortical oocytes (CAO), early vitellogenic oocytes (EVO), late/mature oocytes (LO), spermatogonia (SG), spermatocytes (SC), and spermatids (ST) were shown in (B,C). Asterisks in the figures were regarded as statistical differences between the exposure groups and control group (Tukey: * p < 0.05).

Figure 3

(A) Comparison of forward sperm motility movement parameters between exposure and control groups. VAP, average path speed; VSL, Line motion speed; VCL, curve motion speed (mean ± SEM, n = 10). (B) Schematic representation of sperm motility patterns according to a computer-assisted sperm analysis system. Asterisks in the figures were regarded as statistical differences between the exposure groups and control group (Tukey: * p < 0.05).

Figure 4

(A) Mean swimming speed of female and male zebrafish after exposure for 21 days (mean ± SEM, n = 16). Cumulative swimming distance of female (B) and male (C) zebrafish after exposure to TEB for 21 days (mean ± SEM, n = 16). Social behaviors of females and males (mean ± SEM, n = 16). (D) numbers of contacts, (E) contact time, F-F, female and female; M-M, male and male; F-M, female and male. (F) Real-time pictures of behavior detection. Asterisks in the figures were regarded as statistical differences between the exposure groups and control group (Tukey: * p < 0.05).

Figure 5

Influence of TEB on the levels of E2 and T in female (A) and male (B) zebrafish (mean ± SEM, five fish per replicate, 4 replicates). Asterisks in the figures were regarded as statistical differences between the exposure groups and control group (Tukey: * p < 0.05).

Figure 6

Heat map of gene expressions associated with social behavior and HPG axis of female (A) and male (B) zebrafish (mean ± SEM, n = 6), and the values in the result were derived from the fold change of gene expression converted by a log 2 as the bottom.