Combined Effects of Heat and Cd²⁺ Stress on Growth, Physiology, and Transcriptomic Responses in Sipunculus nudus

Heat and Cd²⁺ stress are major environmental challenges for marine benthic invertebrates. This study examined their combined effects on growth, physiology, and transcriptomic responses in the peanut worm (Sipunculus nudus). After 30 days, Cd²⁺ reduced survival at 26 °C without significantly affecting growth, whereas at 32 °C, both survival and growth declined with increasing Cd²⁺ concentration, indicating that heat stress exacerbates Cd²⁺ toxicity. Cd accumulation increased with exposure concentration but was not affected by temperature. Heat stress increased immune (AKP) and antioxidant (SOD, CAT) enzyme activities, although significant increases in SOD and CAT were observed only under Cd²⁺ exposure. AKP activity rose at low Cd²⁺ concentrations and fell at high Cd²⁺ concentrations at 26 °C, whereas no significant difference occurred at 32 °C between 0 and 0.25 mg/L Cd²⁺. At the same temperature, SOD and CAT activities were significantly higher under high Cd²⁺ exposure than under low Cd²⁺ exposure. Transcriptome analysis showed that Cd²⁺ exposure activated longevity-related pathways, protein processing, and translation initiation. Heat stress activated Jak-STAT signaling and endoplasmic reticulum protein processing while inhibiting the ribosome pathway. Under combined stress, pathways related to xenobiotic metabolism, nutrient digestion and absorption, and amino acid derivative metabolism were broadly suppressed. These results highlight that heat stress exacerbates Cd²⁺ toxicity, affecting growth, enzyme activity, and transcriptomic responses, and provide insights into the adaptive strategies of marine benthic organisms under the combined pressures of climate change and heavy metal pollution.