Institute of Chemistry and Biology of the Marine Environment, Universität of Oldenburg, 26111 Oldenburg, Germany.
In giant danio, Danio malabaricus, (Order Ostariophysi), the characteristic fright reaction is produced by introducing the alarm substance (Schreckstoff), which was extracted from the skin of adult fish. It is known that when the skin of an stariophysian fish is damaged, alarm substace cells are broken and the alarm substance is released. Nearby conspecifics smell the alarm substance and show a fright reaction according to their stimuli. It is therefore very important to determine and confirm the fright reaction definitely to prove the chemical nature of alarm substance in aquatic animal. After introducing the alarm substances into the test aquarium, positive reaction is achieved immediately. The fish tested swim excitedly and move toward the tank bottom. The main purpose of this work is to study the chemical nature of the alarm pheromone in D. malabaricus. We were able to isolate and identify the alarm substance present in the skin of giant danio. Isolation method included extraction, and separation methods was achieved by ultrafiltraction, reversed-phase high-performance liquid chromatography (RP-HPLC) and gel filtration chromatography (GFC). Identification methods consisted of the UV/VIS, 1H-, 13C-, 1H-13C COSY- NMR experiments and laser desorption/ionization mass spectroscopy (LD/IMS). Comparison of biological activity and spectral data corresponding to the natural substance and proposed authentic sample verified that 7-hydroxybiopterin is the alarm substance. The amount for response was determined as 0.05 mg.