Measuring mutagenicity in ecotoxicology: A case study of Cd exposure in Chironomus riparius

Abstract

Existing mutagenicity tests for metazoans lack the direct observation of enhanced germline mutation rates after exposure to anthropogenic substances, therefore being inefficient. Cadmium (Cd) is a metal described as a mutagen in mammalian cells and listed as a group 1 carcinogenic and mutagenic substance. But Cd mutagenesis mechanism is not yet clear. Therefore, in the present study, we propose a method coupling short-term mutation accumulation (MA) lines with subsequent whole genome sequencing (WGS) and a dedicated data analysis pipeline to investigate if chronic Cd exposure on Chironomus riparius can alter the rate at which de novo point mutations appear. Results show that Cd exposure did not affect the basal germline mutation rate nor the mutational spectrum in C. riparius, thereby arguing that exposed organisms might experience a range of other toxic effects before any mutagenic effect may occur. We show that it is possible to establish a practical and easily implemented pipeline to rapidly detect germ cell mutagens in a metazoan test organism. Furthermore, our data implicate that it is questionable to transfer mutagenicity assessments based on in vitro methods to complex metazoans.

Temperature dependence of spontaneous mutation rates

“…Significantly exceeding mean µ estimates at different temperatures, we detected five so-called mutator lines (see above and Supplemental Table S2 ). The spontaneous occurrence of mutator lines is a known phenomenon for MAL experiments ( Haag-Liautard et al 2008 ; Schrider et al 2013 ) and has also been previously observed in C. riparius ( Doria et al 2021 ). As reported by Doria et al (2021) and contrasting with the observation in Drosophila ( Schrider et al 2013 ), mutator alleles most probably arose along the course of our experiment because all other lines deriving from the same ancestral parents produced offspring with normal rates.…”

“…The spontaneous occurrence of mutator lines is a known phenomenon for MAL experiments ( Haag-Liautard et al 2008 ; Schrider et al 2013 ) and has also been previously observed in C. riparius ( Doria et al 2021 ). As reported by Doria et al (2021) and contrasting with the observation in Drosophila ( Schrider et al 2013 ), mutator alleles most probably arose along the course of our experiment because all other lines deriving from the same ancestral parents produced offspring with normal rates. We can thus rule out a natural background to cause the extraordinary increase in mutation rates.…”

“…Finally, the combination of the two mutation detection approaches gave us the most robust and comprehensive set of de novo mutation candidates, all of which were visually curated in the Integrative Genomics Viewer (IGV) (v2.6.3) ( Thorvaldsdottir et al 2013 ), excluding SNMs in the 23°C mutator lines (see below). To account for the possibility that mutator alleles can arise and become fixed within a single MAL, as has been shown in other mutation accumulation studies before ( Schrider et al 2013 , Doria et al 2021 ), we set a conservative threshold of a fourfold increase in mutation rates compared to mutation rates of the same temperature treatment to define a so-called mutator line. We tested for random distribution of MALs with zero de novo mutations performing a χ 2 test with degrees of freedom df = 5.…”

Detecting Cd adaptation footprint in C. riparius with a multi-genomic approach

“…Regarding the DNA time-series investigation, it is important to notice that the microevolution observation of allele frequency changes that occurred over time within the replicates, fundamentally occurred by selection since gene flow, genetic drift and elevated mutation rates (Doria et al, 2021) can be excluded. Hence, it is remarkable that although the final lists underlined allele frequency changes in genes unique to each treatment, those different selection targets still converged into two shared biological functions.…”

“…It can promote oxidative stress and negatively affect immune, inflammatory and reproductive endpoints, being one of the most toxic metals present in aquatic environments (Blickley et al, 2014; Doria and Pfenninger, 2021; Martín-Folgar and Martínez-Guitarte, 2019). But although, it is listed as a carcinogenic and mutagenic substance (Wang et al, 2019), it was recently demonstrated that it does not affect germ cell mutation rates in C. riparius (Doria et al, 2021)…”