Characterizing the bioluminescence of the Hhumboldt squid, dosidicus gigas (d'Orbigny, 1835): One of the largest luminescent animals in the world

Abstract

Bioluminescence is found in a number of cephalopods, such as Watasenia scintillans and Sthenoteuthis oualaniensis; however, many species remain poorly studied, including the Humboldt squid, Dosidicus gigas. This is the largest member of the Ommastrephidae family and grows to 2 m in length, making it one of the largest luminescent animals ever observed. Humboldt squid have small photophores all over their body that emit a brilliant blue luminescence. Using lyophilized photophores from squid caught off the coast of Chile, experiments were conducted to isolate the luciferin and protein involved in its bioluminescence. Methanolic extracts of the photophores were shown to contain dehydrocoelenterazine, and a membrane-bound photoprotein was shown to be involved. This photoprotein was purified using ion exchange chromatography, and SDS-PAGE showed a clean band of approximately 60 kDa. The excised band was analyzed by LC/MS, and the obtained data were compared against the transcriptome data of D. gigas, allowing us to find two gene products which displayed high coverage (>80%), the enzymes symplectin and vanin-2, which potentially associate with light emission process in this organism. Finally, the purified photoprotein was shown to emit a blue light (470 nm) in the presence of dehydrocoelenterazine. Article Note: These authors contributed equally to this work. CAPTION(S): Figure S1. Light emission profile recorded in a luminometer over 60s after addition of dehydrocoelenterazine to crude enzyme extract dissolved in buffer. Dehydrocoelenterazine final concentration 2.5 mm, crude extract 50 [MU]L (1.0 [MU]g protein [MU]L-1), final volume 1000 [MU]L, Tris-HCl buffer (pH 8.5, 50 mm, 0.4 m NaCl). Figure S2. Light intensity over time for different volumes of the crude extracts containing D. gigas photoprotein (left) along with the integrated plots of light intensity for each volume (right). Reactions were carried out using 10 [MU]L of dehydrocoelenterazine, variable volumes (50, 100, 250 and 500 [MU]L) of crude enzyme extract (1.2 [MU]g protein [MU]L-1) in Tris-HCl buffer (pH 8.5, 50 mm, 0.4 m NaCl), final volume 1.0 mL, dehydrocoelenterazine final concentration 2.5 mm. Dehydrocoelenterazine final concentration 2.5 mm, crude extract 50 [MU]L (1.0 [MU]g protein [MU]L-1), final volume 1000 [MU]L, Tris-HCl buffer (pH 8.5, 50 mm, 0.4 m NaCl). Figure S3. Influence of different pH values on the integrated light emission assays. Reactions were carried out by using 50 [MU]L of the crude extract (1.0 [MU]g protein [MU]L-1), 850 [MU]L of the buffer to be tested (pH 4.4, 50 mm acetate; pH 5.4, 50 mm acetate; pH 6.5, 50 mm phosphate; pH 7.5, 50 mm phosphate; pH 8.5, 50 mm Tris-HCl; pH 9.0, 50 mm Tris-HCl), 50 [MU]L of catalase solution (2 mg mL-1) and 50 [MU]L of 5% H2O2, final volume of 1000 [MU]L. Figure S4. Effect of different salts on the solubilisation of D. gigas photoprotein in the crude extracts. Four D. gigas extracts (1.0 [MU]g protein [MU]L-1) were centrifuged at 20 000 g for 10 min at 4[degrees]C and the supernatants were discarded. The obtained pellets were resuspended in 2.0 mL sodium phosphate buffer pH 7.0, 50 mm, with each tube containing a different salt to be tested (1 m KCl, 1 m NaCl, 1 m NaClO4 and 1 m KI) and those extracts were kept under gentle shaking at 4[degrees]C for 1 h. Following this, the homogenates were centrifuged at 20 000 g for 10 min at 4[degrees]C, and the supernatants containing the solubilized protein had their light activity tested by adding dehydrocoelenterazine (final concentration 2.5 mm). Figure S5. Amino acid sequence of symplectin-like enzymes of D. gigas identified by cross analysis of transcriptome and MS data. Figure S6. Influence of different concentrations of Na+ and K+ Influence of different concentrations of Na+ and K+ ions added to the crude extract on the integrated light intensity. The in vitro light emission was examined with 50 [MU]L of crude extract (1.0 [MU]g protein [MU]L-1), 850 [MU]L of Tris-HCl buffer (pH 8.5, 50 mm, 0.4 m NaCl) containing either NaCl/KCl in the following final concentrations (0.4 m, 0.5 m and 0.6 m), 50 [MU]L of catalase solution (2 mg mL-1) and 50 [MU]L of 5% H2O2, final volume of 1000 [MU]L. Byline: Gabriela A. Galeazzo, Jeremy D. Mirza, Felipe A. Dorr, Ernani Pinto, Cassius V. Stevani, Karin B. Lohrmann, Anderson G. Oliveira