| 其他摘要 | Chemical alarm substances play an important ecological role in aquatic predation systems. Aquatic animals exploit water-borne chemical signals to assess the risk of predation and respond with avoidance or escape behavior. Individual fish release alarm substances from their skin when they are attacked by predators. Conspecific individuals use specific chemoreceptors to recognize predation signals, which trigger instinctive behavioral responses such as darting, avoidance and freezing. The chemical alarm system of fish has profound implications for the survival and adaptability of populations. In fish studies, the phenomenon of anti-predatory behavior and physiological response induced by alarm substances has been demonstrated. However, the chemical nature of the alarm substances and their function mechanisms remain enigmatic. Zebrafish (Danio rerio) have been shown to exhibit potent and diverse anti-predatory behavior in
response to alarm substances released by conspecifics. As a classical model for vertebrate neurobiological and marine ecotoxicological research, exploring the chemical molecular structure of the alarm substance that causes anti-predatory behavior in zebrafish and its mechanism can advance the quantification of vertebrate stress behavioral responses and also in-depth understanding of the behavioral and physiological mechanisms of marine swimming animals.
Zebrafish is the subject of the study in the thesis. We focused on the bioassay-guided isolation of alarm substances, the elucidation of their chemical molecular structures, relationship between the concentration threshold of alarm substances and behavioral responses, and the physiological response and its mechanism in zebrafish. The present work is innovative, and several experimental methods have been developed and validated in this study. First, at the beginning of the study, we assessed the alarm behavior of zebrafish using a novel tank diving test paradigm and screened for innate behavioral responses induced by alarm substances. The semi-quantitative evaluation of the typical behavioral phenotypes of alarm substances extracted from skin revealed that erratic movements, freezing, bottom and latency duration are significant behavioral indexes. Second, a UPLC-TOF-MS method was developed for simultaneous determination of six steroid hormones (cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone and 17-hydroxyprogesterone) in zebrafish. This analytical method has been used for the determination of cortisol in the whole body of zebrafish after acute exposure of alarm substances. The method is highly sensitive, robust and accurate, and which provides technical support for the subsequent accurate and efficient evaluation of the physiological response to stress in zebrafish individuals. The method overcomes the shortcomings and drawbacks of the established enzyme immunoassay, which is prone to false positives. The developed and validated method supports the subsequent accurate and efficient evaluation of the physiological response to stress in zebrafish individuals. Third, a quantitative analytical method was developed to accurately analyze the alarm compounds obtained by isolation, and the concentrations of the two compounds in fish skin extracts were accurately determined. Collectively, the above methods have been developed and validated in zebrafish research, providing a scientific foundation for the study of the relationships among alarm substance, behavioral response, and physiological response quantitatively.
The conclusions of this thesis can be drawn as follows:
1. Isolation and identification of two oxysterol sulfates from zebrafish skin extracts, one novel compound trivial named 5DS and one known compound 5α-cyprinol sulfate (5CS).
Phase extraction of zebrafish skin extracts obtains petroleum ether phase, ethyl acetate phase, and n-butanol phase. The three extraction phases were used as stimuli for alarm behavior assays, respectively. The results showed that the n-butanol phase extract could replicate the behavioral response of the crude skin extract of zebrafish. The n-butanol phase was further purified by liquid chromatography, column chromatography, and thin-layer chromatography to obtain two compounds. Elucidation of the chemical structure and relative configuration of compounds by spectroscopic and spectrometric techniques such as NMR and high-resolution mass spectrometry. Of the two compounds identified in the n-butanol phase, one is the novel compound 24-methyl-5α-cholestane-3α,7α,12α,24,28-pentahydroxy 28-sulfate, trivially named 5DS, and the other is the known compound 5α-cyprinol sulfate, denoted as 5CS.
2. The compounds 5CS and 5DS can cause behavioral responses with concentration thresholds of 10–10 M and 10–12 M, respectively.
Different concentrations of compound 5CS and 5DS stimulating solutions were prepared, and the movement trajectories of zebrafish under those stimulating solutions were recorded and analyzed. The results showed that both compounds 5CS and 5DS were able to elicit an alarm behavioral response in zebrafish, and that the alarm behavioral response in zebrafish did not show a linear correlation with the concentration of the alarm substance. There was a concentration threshold for the action of the alarm pheromone, which was 10–10 M for 5CS and 10–12 M for 5DS. Compound 5DS shows a two-orders of magnitude lower than compound 5CS.
3. The alarm behavioral responses of zebrafish stimulated by the compounds 5CS and 5DS showed significant sex differences.
When exposed to compound 5CS, female zebrafish showed more pronounced erratic movements, while males showed more freezing and a significant increase in the latency to the top. In contrast, when exposed to compound 5DS, females generally performed better than males in all four alarm behavior responses. In addition, compounds 5CS and 5DS have different propensities and sex differences for triggering different alarm behavior phenotypes in zebrafish. In female zebrafish, compound 5CS is more likely to induce erratic movements, while compound 5DS tends to trigger freezing behavior. For male zebrafish, the alarm response was dominated by 5DS at low concentrations (10–12 M), showing significantly higher exposure effects for compound 5DS than compound 5CS for erratic movements, freezing and latency to the top. While at higher concentrations (10–10 M), all three behavioral responses showed compound 5CS was significantly more effective than compound 5DS, when it was mainly 5CS
that acted.
4. The acute exposure to compounds 5CS and 5DS elevated cortisol in zebrafish with thresholds of 10–8 M and 10–10 M respectively, and both compounds showed significant sex differences.
When compound 5CS was used as a stimulant, cortisol levels increased with increasing concentrations of compound 5CS in both female and male fish. This pattern was maintained in females when compound 5DS was used as the stimulant. In males, cortisol levels rose sharply to a maximum with increasing concentrations of compound 5DS and then fell rapidly when concentrations continued to rise. In conclusion, stimulation with compound 5CS resulted in generally higher cortisol levels in females than in males overall, whereas stimulation with compound 5DS resulted in generally higher cortisol levels in males than in females. In addition, cortisol levels in female zebrafish showed a significant increase at concentrations of 10–8 M for both compounds. In contrast, male zebrafish showed a significant increase at concentrations of 10–10 M for both compounds, indicating that male zebrafish are more sensitive to the perception of stress. Except for 10–8 M, the content of cortisol in male zebrafish caused by compound 5DS was significantly higher than that of compound 5CS, the cortisol levels in zebrafish stimulated by compound 5CS were significantly higher than those stimulated by compound 5DS at all other concentrations. And this pattern was shown in both female and male zebrafish.
5. Acute exposure to compounds 5CS and 5DS, zebrafish show metabolic pathway differences with regard to sex.
Acute exposure to compounds 5CS and 5DS can both affect zebrafish metabolism. For compound 5CS, PCA and PLS-DA analyses showed that zebrafish metabolites varied more between treatment groups at different concentrations. While for compound 5DS, changes in concentration did not have a significant effect on the zebrafish metabolome. By OPLS-DA and S-plot analysis, it can be concluded that the number of differential metabolites obtained when compound 5CS acts is greater than that of compound 5DS. The results of the metabolic pathway analysis showed that compound 5CS affected more metabolic pathways in zebrafish than compound 5DS. The top five metabolic pathways affected by 5CS were the same in females and males, while there were gender differences in the effects of compound 5DS. The effects of compound 5CS exposure on metabolic pathways in female and male fish involved 16 and 15 pathways, respectively, with the top five most influential pathways all being linoleic acid metabolism, pentose phosphate pathway, valine, leucine and isoleucine biosynthesis, purine metabolism and one carbon pool by folate. Exposure to compound 5DS affected four and five metabolic pathways in female and male fish, respectively, including alpha-linolenic acid metabolism, purine metabolism, arachidonic acid metabolism and biosynthesis of unsaturated fatty acids. The difference is that for female fish, the most important effect is on the metabolism of alpha-linolenic acid, while for male fish the most important effect is on the metabolism of taurine and hypotaurine. |
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