Pollution-Driven Selection in a Non-Biting Midge: Genome-Wide Responses to Bacillus thuringiensis israelensis and Copper.

Riparian ecosystems are vital interfaces between aquatic and terrestrial environments but are increasingly impacted by anthropogenic pollution. In these systems, merolimnic insects serve as crucial ecological links, occupying aquatic habitats as larvae and terrestrial environments as adults, thus being an essential food source in both. Consequently, pollutant exposure during the aquatic larval stage can have cascading effects across ecosystem boundaries. While the ecological consequences of such exposure are well documented, the evolutionary potential of merolimnic insects to adapt to chronic pollution remains poorly understood. To address this, we previously conducted a selection experiment exposing populations of the non-biting midge Chironomus riparius to the mosquito larvicide Bacillus thuringiensis israelensis (Bti) or heavy metal copper over approximately eight generations, which revealed only limited evidence of consistent phenotypic adaptation. Here we use whole-genome sequencing of these populations to assess their genomic responses to chronic pollutant exposure. Despite similar phenotypic sensitivity in pre-exposed and naïve populations, we detected distinct stressor-specific genomic responses. Copper exposure induced a significant genome-wide reduction in nucleotide diversity and evidence of selection-driven allele frequency changes, while Bti exposure was associated with heterogeneous, replicate-specific shifts, potentially reflecting drift or selection on multiple redundant pathways. Functional enrichment analyses indicated early-stage adaptation: immune- and apoptosis-related pathways were enriched under Bti, while metal detoxification and DNA repair pathways were enriched under copper, highlighting distinct adaptive mechanisms despite weak genome-wide signals of selection. Our findings demonstrate that Evolve and Resequencing approaches enable the detection of early genomic signals of adaptation even when phenotypic change is subtle or absent, offering a powerful framework for studying evolutionary responses to environmental pollution.