Efficiency of calcein tagging on juveniles of the sea urchins Diadema africanum and Paracentrotus lividus

Abstract

We evaluated the effectiveness of chemical tagging with the fluorescent marker calcein for two key species of herbivorous sea urchins, Diadema africanum and Paracentrotus lividus, to facilitate medium-and long-term ecological experiments. In total, 98 juveniles of D. africanum and 98 P. lividus were tagged with this fluorescent marker, with 12 combinations of different tagging techniques (chemical bath or injection), concentrations of calcein (2, 10 and 20 mg Á l À1 ), and soaking times (2, 4 and 24 h). Respective control treatments were conducted by means of seawater injection and bathing. The success of tagging was assessed after a month of feeding individuals ad libitum with the algae Dictyota sp. Sea urchins were dissected and their Aristotle's lanterns cleaned with 10% sodium hypochlorite to examine these structures under UV light using a binocular microscope. Each species was evaluated in terms of survival, percentage of tagged individuals and intensity of the resulting tag. The results showed that the method of soaking individuals of both species for 24 h in concentrations of calcein of 10 and 20 mg Á l À1 gave the highest percentage survival (100%) and the resulting tags were clearly visible.

Ocean warming and acidification alter the behavioral response to flow of the sea urchin Paracentrotus lividus

“…Animals were transported alive to Belgium and let to acclimate for 12 days (Cohen-Rengifo et al, 2018). At day 3, individuals were soaked during 24 hr in the fluorescent marker calcein (20 mg/L) to tag their skeletal components (Rodríguez, Hernéndez, & Clemente, 2016;Russell & Urbaniak, 2004).…”

Polychrome labeling reveals skeletal triradiate and elongation dynamics and abnormalities in patterning cue-perturbed embryos

“…In more recent years, researchers have expanded the use of these chemicals by administering multiple fluorochromes sequentially to investigate temporal dynamics of growth and healing in bones (O’Brien et al, 2002; Pautke et al, 2007). Although some of these fluorochromes have previously been used individually or sequentially to study growth in adult tests of several sea urchin species, calcein green is the only calcium-binding fluorochrome that has been used to fluorescently label the larval sea urchin skeleton (Ellers and Johnson, 2009; Johnson et al, 2013; Kobayashi and Taki, 1969; Rodríguez et al, 2016; Vidavsky et al, 2014). Here, we optimize protocols for larval skeleton labeling with alizarin red, xylenol orange, tetracycline, and calcein blue and establish polychrome labeling techniques with xylenol orange, calcein green, and calcein blue to gain temporal insight into normal and perturbed larval skeletal patterning.…”