Small heat shock proteins with two alpha-crystallin domains: a new set of proteins in the earthworm Eisenia fetida with differential transcriptional responses to stressors.

Climate change and environmental pollution are two primary challenges facing biodiversity and ecosystem stability. Earthworms are key contributors to soil structure and nutrient cycling, and their molecular stress responses can provide an early indication of soil health impairment. Heat shock proteins are central to the stress response, and small heat shock proteins (sHSPs) are ATP-independent chaperones that limit stress-induced protein aggregation. Because their expression is stress-sensitive, sHSPs are promising molecular markers for soil stress and contributors to thermotolerance. Eisenia fetida, a widely used ecotoxicology model, relies on molecular chaperones like small heat shock proteins (sHSPs) for stress tolerance. We previously characterized sHSPs containing a single α-crystallin domain (ACD) in E. fetida. Here, we report the first identification of sHSPs containing two α-crystallin domains (ACDs) in annelid species. These genes were identified from an E. fetida transcriptome, their domain architecture was defined, and their transcriptional responses were quantified under heat stress, desiccation, and exposure to two pollutants (bisphenol A and endosulfan), including combined exposure with elevated temperature. Double-ACD sHSPs showed stimulus- and time-dependent transcriptional patterns. Moderate heat and desiccation primarily induced late (24 h) upregulation of several sHSP genes, whereas bisphenol A at optimal temperature did not result in significant transcriptional change and endosulfan produced only limited changes under single-stressor exposure. In contrast, combined exposure to endosulfan and elevated temperature triggered a significant upregulation of multiple sHSP genes, consistent with an additive stress effect. These results expand this protein family diversity in annelids and support a staged sHSP response in which structurally distinct sHSPs may contribute to resilience under prolonged or combined environmental stress.