Paracetamol Responses Depend on Temperature, Life Stage, and Genetic Background in Drosophila melanogaster Across Generations

Background: A key obstacle in toxicological research is investigating long-lasting or inherited effects and addressing the potential interaction between exposure and genetic background of the test subject. Such effects can be effectively investigated in short-lived and genetically managed model organisms. Methods: To illustrate the biological complexity of the outcomes of toxicological experiments, we investigated the influence of life stages, sex, and temperatures in a generational study using paracetamol exposure as an example of drug toxicology. To also include genetic background, we used five highly inbred Drosophila melanogaster lines from the Drosophila Genetic Reference Panel (DGRP). First, we assessed acute survival to paracetamol exposure at 19 °C and 25 °C, revealing clear genotype-specific dose–response curves and increased toxicity at lower temperature. We then conducted a generational experiment across three generations, in which flies were exposed during either the larval or adult life stage, and measured starvation tolerance and spontaneous activity to capture direct, intergenerational, and transgenerational effects. Results: Results show that life stage, sex, and genotype each shaped the phenotypic outcome of paracetamol exposure. Adult exposure consistently decreased activity and increased starvation resistance, whereas juvenile exposure produced more variable and sex-dependent responses. Genotype further modulated these patterns, including markedly elevated lethality in one line at high paracetamol concentrations. Conclusions: These findings demonstrate that exposure timing and biological context critically influence toxicological outcomes, underscoring the need for methodological awareness when interpreting generational studies in model organisms and beyond.