Single-Larva RNA Sequencing Identifies Markers of Copper Toxicity and Exposure in Early Mytilus californianus Larvae.

Megan R Hall,Andrew Y Gracey

doi:10.3389/fphys.2021.647482

Abstract

One of the challenges facing efforts to generate molecular biomarkers for toxins is distinguishing between markers that are indicative of exposure and markers that provide evidence of the effects of toxicity. Phenotypic anchoring provides an approach to help segregate markers into these categories based on some phenotypic index of toxicity. Here we leveraged the mussel embryo-larval toxicity assay in which toxicity is estimated by the fraction of larvae that exhibit an abnormal morphology, to isolate subsets of larvae that were abnormal and thus showed evidence of copper-toxicity, versus others that while exposed to copper exhibited normal morphology. Mussel larvae reared under control conditions or in the presence of increasing levels of copper (3–15 μg/L Cu2+) were physically sorted according to whether their morphology was normal or abnormal, and then profiled using RNAseq. Supervised differential expression analysis identified sets of genes whose differential expression was specific to the pools of abnormal larvae versus normal larvae, providing putative markers of copper toxicity versus exposure. Markers of copper exposure and copper-induced abnormality were involved in many of the same pathways, including development, shell formation, cell adhesion, and oxidative stress, yet unique markers were detected in each gene set. Markers of effect appeared to be more resolving between phenotypes at the lower copper concentration, while markers of exposure were informative at both copper concentrations.

Highlights

Heavy metal contamination of freshwater and marine water bodies is a long-recognized problem, especially in urban regions where industrial byproducts are high (Livingstone et al, 1992)
In order to identify sensitive molecular biomarkers of copper exposure, we previously investigated the concentrationresponsive molecular changes associated with copper exposure in the mussel embryo-larval assay by generating expression data from pools of larvae exposed to a range of 10 copper concentrations (Hall et al, 2020)
We have identified robust transcriptional markers of copper exposure and effect in M. californianus larvae that could be used to improve the sensitivity and objectivity of bivalve embryo water quality assays for copper

Summary

Introduction

Heavy metal contamination of freshwater and marine water bodies is a long-recognized problem, especially in urban regions where industrial byproducts are high (Livingstone et al, 1992). Single-Larva Markers Copper Exposure Toxicity (hpf), when normal larvae reach the D-veliger stage. At this point, abnormal animals exhibit gross morphological deformities, including velum protrusions, misshapen shells, and failure to form shells (His et al, 1997; E50 Committee, 2013). Abnormal animals exhibit gross morphological deformities, including velum protrusions, misshapen shells, and failure to form shells (His et al, 1997; E50 Committee, 2013) This test is typically conducted as a dose response assay in which larvae are exposed to a range of concentrations and an effective concentration at which 50% of the population becomes abnormal (EC50) is determined (E50 Committee, 2013; EPA, 2016). The normal development assay is relatively coarse and fails to capture more nuanced and sensitive physiological responses to chemical exposure or toxicity

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Discussion

Conclusion