Guppy gene expression dynamics: effects of sex, reproductive status, feeding, and temperature on reproductive function, methylation, and appetite

Azari, Sepideh (2025) Guppy gene expression dynamics: effects of sex, reproductive status, feeding, and temperature on reproductive function, methylation, and appetite. Masters thesis, Memorial University of Newfoundland.

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Abstract

Introduction: This thesis examines the dynamics of gene expression in guppy fish (Poecilia reticulata) under various growth conditions, diets, and temperatures. Understanding the molecular processes underlying methylation, reproduction, and appetite regulation in these fish is the goal of this research. The process by which genetic information is transformed into useful products like proteins is known as gene expression. Guppy reproduction is highly impacted by elements like diet and temperature. This work investigates the effects of these factors on gene expression. Methodology: Male and female guppies (Poecilia reticulata) were housed in 20L aquariums with filtered water at 25°C under a 12-hour light/dark cycle. Fish were acclimated for a week and fed daily with Fancy Guppy meal. For gene expression analysis, fish were divided into experimental groups: 1) fasting vs. feeding for 14 days, 2) temperature effects (25°C, 32°C, and 18°C). Tissues (brain, liver, intestine) were collected post-mortem for RNA extraction using the GeneJET RNA Purification Kit. Total RNA was reverse transcribed using the SensiFAST cDNA Synthesis Kit. Specific gene expression was analyzed via qPCR, and primer design was based on Poecilia reticulata sequences. Data were analyzed using the 2-ΔΔCt method, normalized to reference genes GAPDH, EF-1α, and β-actin. Statistical analyses included ANOVA and non-parametric tests with a significance level of P<0.05. Results: A total of 45 guppies were examined, including 33 females and 12 males, with females classified into three reproductive stages. Analysis of brain gene expression showed no significant differences in orexin (OX) or NPY levels between groups, but Kiss2 expression was higher in females with late-stage embryos, while AVP was elevated in males. Intestinal and liver genes expression showed no significant changes. In fasting experiments, females consumed more food than males, with brain orexin in fast male and hepatic dnmt3b expression in fed male being significantly higher. Gnrh3 and npvf expression in fasted male and IT expression in fed female were higher. Temperature experiments revealed that food intake increased at higher temperatures, and npvf and Orexin levels were elevated at 32°C and 18°C in males. However, no significant changes were observed in epigenetic-related genes in response to temperature variations. Conclusion: This study demonstrates how environmental elements like temperature and diet can have a substantial impact on guppy gene expression and phenotypic adaptation. The results of this study advance our knowledge of the molecular mechanisms underlying living things' flexibility and adaptation to environmental changes and may find use in aquaculture and conservation.

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