ABS 028: Non-Additive Effects on Gene Expression in Daphnia magna Under Toxicological Multi-Stressor Conditions

Katherine Gilboy ¹ , Neil McAdams ¹ , Michael Pfrender ¹

¹ University of Notre Dame

Van Wickle (2025) Volume 1, ABS 028

Introduction: With the rise of microplastics and various synthetic chemicals permeating our water and food supply, there is an increasing concern on how these chemicals affect human health. Two of these prominent toxic substances include cadmium and perfluorooctane sulfonate (PFOS). Studies have shown increased levels of cadmium and perfluorooctane sulfonate to disrupt vital metabolic pathways in the human body and perfluorooctane sulfonate has been linked with disorders such as depression. In Daphnia magna, survival and reproductive traits such as clutch size, reproductive timing, metabolic functions, and lifespan have been shown to be negatively impacted. Cadmium and perfluorooctane sulfonate have been observed to have synergistic effects, amplifying one another's effects. Conversely, sodium chloride has been shown to counteract some of the effects of perfluorooctane sulfonate. However, how the combined impacts of cadmium, perfluorooctane sulfonate, and sodium chloride on gene expression is poorly understood. In this study, three generations of Daphnia magna were propagated, with the fourth generation being subjected to distinct environmental conditions containing cadmium, perfluorooctane sulfonate, and sodium chloride. The RNA was extracted, sequenced, and processed to undergo differential gene analysis. We proposed a non-additive model of gene expression where a greater number of genes would be differentially expressed than the combination of single stressor genes. Compared to the genes affected by a single stressor, 1186 additional genes were differentially expressed in response to the tri-stressor condition. Additionally, 744 more genes were differentially expressed in the tri-stressor condition than in the paired stressor conditions. The GO terms conducted were consistent with stress response in each single stressor based on literature findings, but each condition had different metabolic functions that were significantly differentially expressed. This study aims to better understand transcriptional responses to different combinations of stressors, providing insights into broader implications of toxicological effect on gene expression.

Methods: The organisms were then propagated for three generations and the first clutch of each generation was discarded. The third generation was the exposure generation with eight testing condition consisted of the control, individual exposures of NaCl, PFOS, and cadmium, and combined exposures of NaCl+PFOS, NaCl+cadmium, PFOS+cadmium, and NaCl+PFOS+cadmium. The exposures lasted 24 hours. The individuals were then harvested. RNA was then extracted, sequenced and into cDNA libraries. Fastqc was run to see the quality of the raw reads. The reads were then mapped, indexed, then sorted. Differential gene expression and GO term analysis were conducted.

Results: The number of genes being differentially expressed dramatically increases when more than one stressor is added. In the single stressor condition there were 119 genes compared to 91 in the paired condition. However, in the single stressor versus all stressor comparison, there were 1189 extra genes differentially expressed. In the paired versus all comparison, there were 744 genes differentially expressed. NaCl and PFOS showed similar impacted GO terms whereas Cd and All condition showed similar GO terms. There were no GO terms that overlapped in all 4 conditions, but some that overlapped in three of the four conditions.

Discussion: The All, PFOS+NaCl, and Cd+PFOS showed synergistic effects, showing a non-additive model. In the NaCl+Cd condition, there were less genes differentially expressed than the additive model, demonstrating an antagonistic effect. The GO terms revealed that each stressor impacted different types of metabolic pathways. This indicates that there is a different set of genes that respond to multiple stressors, changing the total amount of differentially expressed genes, fitting a non-additive model. The response being shown was stressor specific. In future analysis, we hope to identify specific genes in order to understand their role in stress response and the different pathways involved.