Gentle protocol to prepare aposymbiotic corals which retained comparable physiological and biochemical performances to their symbiotic counterparts by incubation in seawater only (for Stylophora) or with additional feeding of a nutrient cocktail containing glycerol, vitamins, and a host mimic FAA mixture (for Isopora). Bleaching coral by menthol, as indicated in Fig. 1B, occurred in a significant dose-dependent manner. However, because continuous incubation always caused high mortality, a repeated 8: 16-h menthol (treatment): ASW (resting) treatment cycle was essential for the success of the protocol (Fig. 2). Menthol is a compound known to act on a variety of different membrane receptors, including the transient receptor potential (TRP)M8, TRPA1, and other ionotrophic receptors [39]. The binding of menthol to TRPM8 results in an increase in intracellular Ca2+ concentrations and causes a cold sensation in MedChemExpress LED 209 vertebrates 1326631 [40?3]. Menthol was also found to cause antinoci-ceptive and local anesthetic effects in neuronal and skeletal muscles via blocking voltage-operated sodium channels [44]. Menthol is also known to cause many adverse effects to plants, including photoinhibition [45]. In Symbiodinium-associated corals, the mechanism underpinning menthol-induced coral bleaching is not clear. However, based on two different Symbiodinium-releasing modes (ejecting the alga in a cloudy suspension by Isopora and releasing digested alga by Stylophora), the bleaching mechanism might be attributed to Ca2+-triggered exocytosis as described by Pang and Sudhof [46] and/or photoinhibition in Symbiodinium. We ?have no information about Ca2+ movements in the coral host during menthol treatment, but a preliminary study indicated that menthol might inhibit Symbiodinium photosynthesis II activity in the millimolar range (4-h IC50 of 0.72,1.96 mM) which was at a similar level that caused coral bleaching (unpublished data). Further studies are needed to clarify the mechanism of mentholinduced coral bleaching.Table 2. Contents of free amino acids and activities of malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) in tissue homogenates of symbiotic and bleached Isopora palifera with or without nutrient supplementation.TreatmentMDHGDHFree amino acids Total Essential(nmole NAD(P) min Symbiotic control Apo-symbiotic host Apo-symbiotic host fed nutrient A Apo-symbiotic host fed nutrient B 77618a (14) 8666 (9) 109626 (5) 4269 (11) F3,35 = 2.331 P.0.a a a+mg) 4066a (13) 2064 (9)ab a b(pmole mgb a) 103615a (9) 5868b (11) 94611a (5) 8068ab (11) F3,32 = 3.264 P,0.385643a (9) 213621 (11) 372629 (5) 281634 (11) F3,32 = 5.864 P,0.b(5)4165 (8) F3,31 = 3.292 P,0.Essential amino acids followed the definition applied to the sea 4 IBP anemone Aiptasia pulchella [19]. Enzyme activities were determined as the amount of NAD(P)H (in nmol) converted to NAD(P) by 1 mg of protein in 1 min. Nutrient compositions of A and B are described in “Materials and Methods” and Table 1. Numbers in parentheses represent the number of colony replicates, and means followed by the same letter do not significantly differ at p = 0.05 (Fisher’s least significance difference test). Data are the mean6S.E. doi:10.1371/journal.pone.0046406.tMenthol-Induced Aposymbiotic Coral PerformanceTable 3. Contents of free amino acids and activities of malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) in tissue homogenates of symbiotic and bleached Stylophora pistillata.TreatmentMDHGDHFree amino acids Tot.Gentle protocol to prepare aposymbiotic corals which retained comparable physiological and biochemical performances to their symbiotic counterparts by incubation in seawater only (for Stylophora) or with additional feeding of a nutrient cocktail containing glycerol, vitamins, and a host mimic FAA mixture (for Isopora). Bleaching coral by menthol, as indicated in Fig. 1B, occurred in a significant dose-dependent manner. However, because continuous incubation always caused high mortality, a repeated 8: 16-h menthol (treatment): ASW (resting) treatment cycle was essential for the success of the protocol (Fig. 2). Menthol is a compound known to act on a variety of different membrane receptors, including the transient receptor potential (TRP)M8, TRPA1, and other ionotrophic receptors [39]. The binding of menthol to TRPM8 results in an increase in intracellular Ca2+ concentrations and causes a cold sensation in vertebrates 1326631 [40?3]. Menthol was also found to cause antinoci-ceptive and local anesthetic effects in neuronal and skeletal muscles via blocking voltage-operated sodium channels [44]. Menthol is also known to cause many adverse effects to plants, including photoinhibition [45]. In Symbiodinium-associated corals, the mechanism underpinning menthol-induced coral bleaching is not clear. However, based on two different Symbiodinium-releasing modes (ejecting the alga in a cloudy suspension by Isopora and releasing digested alga by Stylophora), the bleaching mechanism might be attributed to Ca2+-triggered exocytosis as described by Pang and Sudhof [46] and/or photoinhibition in Symbiodinium. We ?have no information about Ca2+ movements in the coral host during menthol treatment, but a preliminary study indicated that menthol might inhibit Symbiodinium photosynthesis II activity in the millimolar range (4-h IC50 of 0.72,1.96 mM) which was at a similar level that caused coral bleaching (unpublished data). Further studies are needed to clarify the mechanism of mentholinduced coral bleaching.Table 2. Contents of free amino acids and activities of malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) in tissue homogenates of symbiotic and bleached Isopora palifera with or without nutrient supplementation.TreatmentMDHGDHFree amino acids Total Essential(nmole NAD(P) min Symbiotic control Apo-symbiotic host Apo-symbiotic host fed nutrient A Apo-symbiotic host fed nutrient B 77618a (14) 8666 (9) 109626 (5) 4269 (11) F3,35 = 2.331 P.0.a a a+mg) 4066a (13) 2064 (9)ab a b(pmole mgb a) 103615a (9) 5868b (11) 94611a (5) 8068ab (11) F3,32 = 3.264 P,0.385643a (9) 213621 (11) 372629 (5) 281634 (11) F3,32 = 5.864 P,0.b(5)4165 (8) F3,31 = 3.292 P,0.Essential amino acids followed the definition applied to the sea anemone Aiptasia pulchella [19]. Enzyme activities were determined as the amount of NAD(P)H (in nmol) converted to NAD(P) by 1 mg of protein in 1 min. Nutrient compositions of A and B are described in “Materials and Methods” and Table 1. Numbers in parentheses represent the number of colony replicates, and means followed by the same letter do not significantly differ at p = 0.05 (Fisher’s least significance difference test). Data are the mean6S.E. doi:10.1371/journal.pone.0046406.tMenthol-Induced Aposymbiotic Coral PerformanceTable 3. Contents of free amino acids and activities of malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) in tissue homogenates of symbiotic and bleached Stylophora pistillata.TreatmentMDHGDHFree amino acids Tot.