Toxicity of the Lessepsian pufferfish Lagocephalus sceleratus from eastern Mediterranean coasts of Turkey and species identification by rapid PCR amplification.
Pufferfish, Lagocephalus sceleratus, L. suezensis and L. spadiceus, are widely distributed Lessepsian species along the Turkish and other eastern Mediterranean coasts. L. sceleratus is considered one of the worst invasive species in the Mediterranean Sea and recently caused human intoxication by accidental consumption. Therefore, methods to assess pufferfish toxicity and accurately identify toxic pufferfish are important for food safety. Firstly, 20 specimens of L. sceleratus were collected from the eastern Mediterranean coasts of Turkey. Tetrodotoxin (TTX) content was determined among different tissues (muscle, liver, gonad, kidney, intestine and skin) by liquid chromatography-tandem mass spectrometry. All of the specimens contained TTX. The highest TTX concentration was detected in the ovary (80.0 µg TTX/g), followed by the intestine (48.8 µg TTX/g), kidney (34.0 µg TTX/g) and liver (25.4 µg TTX/g). The flesh and testis contained detectable amounts of TTX (3.4 µg TTX/g and 2.6 µg TTX/g, respectively). TTX analogs were also detected in the tissues, with 4,9-anhydro-5,6,11-trideoxyTTX and 5,6,11-trideoxyTTX being the major analog. A species-specific PCR method was developed to identify three species of genus Lagocephalus based on the nicotinamide adenine dinucleotide dehydrogenase subunit 2 or cytochrome oxidase subunit I regions of the mitochondrial DNA of L. sceleratus, L. suezensis, and L. spadiceus. The designed primers amplified only DNA of target species and not those of other fish and shellfish species. The species-specific PCR method successfully identified these pufferfish species and could be applied to thermally treated products of the pufferfish with only 1 ng of extracted DNA.