To prepare crude Pontodrilus luciferase and luciferin, live earthworms were rinsed with distilled water and transferred for 24 h to Petri dishes with wet tissue paper moistened with artificial seawater to avoid contamination by their stomach contents when extracting coelomic fluid. All experiments were carried out on ice except for the measurements of light intensity and spectra. Coelomic fluid was extracted as follows: twenty live worms of each species (2.72 g wet weight of P. litoralis and 7.4 g wet weight of P. longissimus) were put on a mortar and stimulated with a pestle to induce exudation of coelomic fluid, and then 10 ml of 50 mM Tris–HCl at pH 7.2 was added. After removing the specimens, the solution was centrifuged at 15,000 × g for 15 min at 4 °C in a TOMY MX-100 high speed refrigerated microcentrifuge (Tomy Seiko, Japan), and the supernatants were collected as the crude extracts. The crude luciferin and luciferase fractions were prepared based on the method described by Bellisario et al.39. In brief, the crude extracts were filtered using a 10 K centrifugal filter device (Merck, Germany); the first flow through was used as a crude luciferin extract, and the retentates on the membranes were collected as crude luciferase extract.

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