Effects of cryopreservation-related processes on the settlement competence of coral larvae

Coral larval settlement constitutes a pivotal bottleneck in reef persistence, restoration efforts, and the development of assisted conservation strategies such as cryopreservation. Beyond survival, the ability of larvae to retain settlement competence following cryopreservation-related stress is essential for the successful application of these approaches. Despite its ecological significance, the mechanistic basis underlying settlement induction remains incompletely understood. Here, we systematically investigate the interactive effects of low temperature, water-soluble chemical cues, and lipid-based treatments on the settlement competence of Pocillopora acuta larvae, with the aim of also exploring the potential of cryopreservation-related stressor as alternative settlement-inducing treatments. Larvae were subjected to controlled chilling regimes, both independently and in combination with a suite of chemical agents across a range of concentrations and exposure durations. Settlement responses were benchmarked against induction by crustose coralline algae (CCA), a well-established natural cue. To disentangle thermal and chemical influences, parallel experiments were conducted under room temperature conditions. In addition, lipid-based delivery systems, including phospholipid and fatty acid liposomes, were evaluated for their potential to enhance settlement competence. Our results reveal that low-temperature exposure supported larval settlement competence at low to intermediate chemical concentrations (0.1-1 M), whereas higher concentrations (2 M) result in rapid loss of viability within 2 h. Settlement success ranged from 11% to 44%, with a peak of 66.6% achieved following exposure to 0.5 M propylene glycol for 2 h. Notably, the combined application of chilling and CCA resulted in reduced settlement relative to either cue alone, suggesting potential antagonistic interactions. Under room temperature conditions, chemical treatments elicited limited and variable settlement responses (≤33%), with methanol showing the highest post-treatment attachment and subsequent development. In contrast, lipid-based treatments failed to induce settlement or metamorphosis. Collectively, these findings demonstrate the remarkable plasticity of larval settlement responses in P. acuta and show that chilling and selected cryopreservation-related treatments do not preclude settlement competence under the conditions tested. Furthermore, some of these treatments may represent promising alternative approaches for inducing settlement under controlled laboratory conditions, and highlights new avenues for enhancing larval settlement for coral reef restoration.