Sublethal oxytetracycline and copper exposure alters rRNA gene copy number, expression, and intragenomic polymorphism...

Ribosomal RNA (rRNA) genes serve as foundational markers for microbial eukaryotic diversity assessment, yet their responses to environmental stressors remain underexplored. This study examined the effects of sublethal oxytetracycline and CuCl₂ on phenotypic and ribotypic traits in the ciliated protists Paramecium bursaria and Euplotes vannus using single-cell quantitative PCR and high-throughput sequencing of 18S rDNA/rRNA. We found that both pollutants inhibited growth, enlarged cell volume, and elevated per-cell rDNA and rRNA copy numbers, but oxytetracycline selectively elevated rRNA: rDNA ratios. These effects increased the variance around established allometric scaling relationships between rDNA copy number and cell volume, without significantly altering the scaling slope. Intragenomic polymorphisms also increased, with the amplicon sequence variant (ASV) number of rRNA transcripts exceeding that of rDNA, and these elevated polymorphisms persisted post-stress relief. Re-analysis of field 18S metabarcoding data from CuCl₂-polluted marine biofilms revealed dose-dependent increases in the number of ASVs per operational taxonomic unit (OTU, defined at a cutoff of 97% sequence identity) in many protistan groups, suggesting that copper-induced mutagenic pressure on ribosomal RNA genes may prevail across diverse protistan taxa. Our findings imply that: (1) when interpreting ASV-based microbial diversity patterns, the potential for ASV richness inflation and elevated per-cell rDNA and rRNA abundances should be recognized as a likely consequence of exposure to environmental pollutants; (2) there is a potential for using the ASV-to-OTU number ratio in assessing environmental stress; and (3) metabarcoding of rRNA likely introduces more artificial ASVs than targeting rDNA. A combination of double metabarcoding of both rDNA and rRNA to identify active microbial members is recommended.