Effective fluorochrome marking of juvenile sea cucumbers for sea ranching and restocking

Dermal spicules (or ‘ossicles’) of cultured sea cucumbers can be fluorescently marked with tetracycline and calcein for sea ranching and restocking but optimal immersion conditions are unknown. Lethal and non-lethal effects, and the efficacy of marking spicules in juvenile sandfish (Holothuria scabra), were examined under different immersion conditions. Fluorescence brightness and the proportion of marked spicules generally increased with concentration and duration of immersion. Frequency of burial (an indicator of stress) in sandfish increased with both fluorochromes at concentrations above 50 mg L^{− 1}. Growth in the two weeks post-marking was unaffected at immersion concentrations of 50 and 100 mg L^{− 1} compared to controls, but appeared inhibited by immersion in solutions of 200 and 400 mg L^{− 1} of tetracycline or calcein. Sequential marking by tetracycline (yellow) and calcein (green), in either order, showed that calcein was deposited in a higher proportion of spicules. Three other fluorochromes with disparate colors, alizarin complexone, calcein blue and xylenol orange, also marked sandfish spicules and expanded the variety of dichromic combinations. Both tetracycline and calcein fluoresced more brightly when juveniles were marked at 26 or 30 °C than at 21 °C, and this low temperature appears also to reduce the proportion of spicules marked by tetracycline. Our findings show that seawater temperature should be regulated for ex situ immersion marking. The behavioral and biological sensitivities of sandfish demand care in administering the fluorochromes. Fluorochrome immersion at 100 mg L^{− 1} for 24 h at ≥26 °C provides a practical compromise between minimizing the fitness of released juveniles and ensuring the efficacy of the markers for studies on the growth and survival of sea cucumbers stocked in the wild.