High Heavy Metal Tolerance of Larvae of Acidic Hot‐Spring Chironomid <i>Chironomus sulfurosus</i> : Toxicity Assay and Transcriptome Analysis

Larvae of the chironomid species Chironomus sulfurosus inhabit freshwater near hot springs, wherein acidic environments promote the leaching of heavy metals from the sediments. In this study, we aimed to characterize the potential of heavy metal tolerance in C. sulfurosus larvae. The median lethal concentrations (LC₅₀) values of heavy metal ions such as Cu²⁺, Pb²⁺, and Cd²⁺ in C. sulfurosus larvae were as follows: 7.52 mM for Cu²⁺, 7.47 mM for Pb²⁺, and 6.06 mM for Cd²⁺, after heavy metal exposure for 24 h. These values were more than two-fold those for Chironomus yoshimatsui larvae (2.62 mM for Cu²⁺, 1.74 mM for Pb²⁺, and 0.70 mM for Cd²⁺), which were collected near nonacidic irrigation canals. These results demonstrate the superior heavy metal tolerance of C. sulfurosus larvae. To explore the molecular mechanisms underlying this tolerance, we performed de novo bulk RNA sequencing analysis of C. sulfurosus larvae exposed to 1.0 mM Cu²⁺, Pb²⁺, and Cd²⁺ for 24 h, compared to a nonexposed control group. Differential transcript expression analysis revealed 2790, 2858, and 4842 upregulated transcripts and 2253, 2577, and 4488 downregulated transcripts in response to Cu²⁺, Pb²⁺, and Cd²⁺ exposure, respectively. Notably, transcripts encoding cuticular-related and Osiris family proteins were commonly involved in tolerance to these heavy metal ions. These results suggest that the highly expressed genes encoding cuticular-related and Osiris family proteins in C. sulfurosus can serve as biomarkers for environmental heavy metal contamination in toxicological assessments.