| The evolution of complex organisms has been driven by interactions between the individuals and their living environment.To understand the evolutionary process,it is critical to unravel the adaptation mechanisms of organisms to environmental cues and changes.Fishes,arguably the most diverse group of vertebrate animals,provide ample opportunities and model systems to decipher the mechanisms whereby vertebrate animals adapt to changes in their environments.We chose zebrafish as our model to study the adaptation of fish to changes in the aquatic environment.We investigated the effects of certain environmental stress factors,including both biological factors and non-biological factors,on fish behaviors.Among biological factors,we first examined the alarm substances,which has been generally recognized as an environmental stress factor that represents predation pressure and,hence,causes anti-predator behaviors.This type of behavior in fish is a complex behavioral process that is likely induced by a combination of active components.We therefore analyzed the components of the embryo extract and skin extract of zebrafish.In addition,we studied another biological stress factor,the social stress.Subsequently,we investigated the effects of certain non-biological environmental factors,including background selection and open fields on fish behavior.Moreover,we examined the effects of how representative non-biological factors influence the anti-predator behaviors induced by alarm substances,further exploring the mechanisms whereby fish adapt to environmental changes.Alarm substances play an important role in the life history of fish and are directly related to the survival of fish.The concept of “alarm substance” was first proposed by a German-Austrian zoologist,Karl von Frisch,who described “schreckstoff” in 1938.Since then,a large number of studies,including a large number of field investigations,have been carried out on fish alarm substances and alarm responses.These studies have shown that alarm substances are likely ubiquitous in fishes.However,a fundamental issue,the chemical nature of alarm substance,has not been solved unequivocally,despite numerous attempts by many researchers to isolate and identify alarm substances.Therefore,the first part of this dissertation focuses on the experimental characterization and discussion of the chemical nature of alarm substance.The behavior responses of fish to alarm substances are important to their fitness.The basic function of anti-predation behavior is to avoid predators,or to reduce the risk of predation,and thus is important for the survival of fish.In addition,the pigment pattern of fish often plays a critical role in avoiding predation.Moreover,pigment patterns play an important role in how fish respond to other environmental stresses.However,current research has been mainly focused on how fish adjust pigment patterns to cope with predation risk in general.Whether changes in pigment patterns affect the anti-predator behavior(or strategy)has not been reported.Therefore,the second part of dissertation is to explore the relationship between pigment patterns and environmental stress response.We exposed zebrafish with two completely different pigment patterns to the same environmental stress stimulation,including biological factors,such as predation pressure and social pressure,and Non-biological factors,such as background selection,open fields.We compared their behaviors under different environmental pressures to reveal the influence of pigment patterns on the response of fish to environmental pressures.In this dissertation study,I first attempted to characterize the chemical nature of the alarm substance.I developed a possibly effective way to isolate and identify candidate components of zebrafish alarm substance by exploring the differences in alarm substance production during different development stages.Subsequently,by comparing two groups of zebrafish with totally different pigment patterns,we examined the influence of pigment patterns on the alarm response,background selection,anxiety,social interaction and other behaviors in zebrafish.To further characterize the alarm substance,we examined extracts of embryos and adult skins of zebrafish.Firstly,we compared the alarm activity present in the extracts of embryos of different development stages,and confirmed the critical time(24 hours post fertilization,hpf)when the embryos start to produce alarm substance.Secondly,we reasoned that embryos at 18 hpf should not contain alarm substances whereas those at 27 hpf should,and therefore,compared the chemical profiles of extracts between 18 hpf and 27 hpf embryos.Through this differential analysis,we screened out that a compound at m/z 330 was present in the 27 hpf embryos but not in the 18 hpf one.We speculated that m/z 330 is possibly a candidate component of the embryonic alarm substance.In the meantime,we fractionated extracts of zebrafish skin into 60 fractions(z1-z60)through liquid chromatography,and subjected each fraction to ultra-high performance liquid chromatography-high resolution mass spectrometry analysis.Based on the results,we grouped the 60 fractions into six pools: Pool1,z1-z6;Pool2,z7-z16;Pool3,z17-z28;Pool4,z29-z37;Pool5,z38-z44;and Pool6,z45-z60.Each pool was tested for its behavioral activity.After three rounds of activity assessment,we determined that the combination of z31 and z37 in pool4 could induce an alarm response in zebrafish.Further analyses indicated that z31 and z37 contained compounds at m/z 301 and m/z 388,respectively.We hypothesize that compounds at m/z 330,301 and 388 are candidate components of alarm substances in zebrafish.This hypothesis needs to be tested through elucidation of the molecular structures and function of these three compounds.To examine the effect of pigment pattern on zebrafish behavior,we observed movement patterns of wild type and nacre type,a mutant with a lighter pigment pattern,under various stress or environmental conditions.When exposed to skin extracts(alarm substance),the nacre fish displayed “bottom” and “freezing” behaviors for longer periods than the wild type.It is possible that “freezing” may be a more effective antipredator strategy when the background and the pigment pattern of nacre matches.In another line of experiments,we compared the light and dark preferences of zebrafish with the two pigment patterns in a black-white box model,and found that nacre individuals preferred the white background in comparison to the wild type.We speculate that the white background matches with the color pattern of the nacre individuals better than the black background,providing a better masking effect.In the third line of experiments,we compared the anxiety behavior and social interaction behavior between the zebrafish of the two pigment types,and found that the zebrafish with two pigment patterns showed significant differences.Although the neuro-ethology mechanisms and the fitness benefits of these behavior patterns remains unclear,our results provide a behavior model and experimental bases for further studies of fish adaptation to environments and selection for pigment patterns in fish and other animals. |
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