Saxitoxin in octopi from Cooke Point, Port Hedland, Western Australia

Full description

Octopus (Abdopus) sp. 5 were collected from a reef flat at Cooke Point, Port Hedland in northern Western Australia. The octopi had a body size of between 5-6 cm and tentacle span of between 20 and 30 cm.Specimens were euthanased by rapid decapitation, stored at -20°C and shipped frozen to the Australian Institute of Marine Science in Townsville. Three legs from each of the four individuals were pooled for toxin extraction. Each of the four samples was extracted with 80% ethanol, homogenised, sonicated three times for 10 minutes, then clarified by centrifugation. This process was repeated two additional times with fresh extraction solvent. For each of the four extracts, the three supernatants generated were pooled, filtered through 0.2 mm nylon filters and lyophilised. Dried extracts were reconstituted in 0.05 M acetic acid and passed through a centrifugal ultra-filter and the ultra filtrates then used for radio-receptor assay and preliminary chemical analysis.Quantitation of STX concentration equivalents (STXeq) with [³H]-STX radio-receptor assays using saxiphilin and voltage gated sodium channel, was performed and octopus extracts were assayed in triplicate for inhibition of STX binding in the sodium channel and saxiphilin assay.All sample extracts which competed with [³H]-STX in the radio-receptor assays were analysed by LC-FLD for STX, decarbamoylSTX, neoSTX, 11-hydroxysulphate STXs (GTX1-6), N-sulfocarbamoyl-11-hydroxy-sulphate STXs (C1-4), hydroxybenzoate STXs and TTX.Identification of suspect toxin peaks was undertaken by mass spectrometric analysis of separated toxins using liquid chromatography-mass spectrometry (LC-MS) performed in single ion monitoring (SIM) mode.
Previous research indicates that Port Hedland on the north-west coast of Australia is a source of marine animals, encompassing a range of crustacea, gastropod and bivalve molluscs, which harbour both tetrodotoxin (TTX) and paralytic shellfish toxins (PSTs). This study was undertaken as part of a renewed investigation into paralytic shellfish toxin distribution in benthic organisms in this region.
This was the first time that saxitoxin had been reported from this family of predatory molluscs and highlights the need for greater public awareness of risks associated with consumption of previously unrecognised vectors of paralytic shellfish poisoning, such as octopus and other benthic feeders.

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Maintenance and Update Frequency: notPlanned

Statement: Statement: Buffers and general reagents were sourced from Sigma (Castle Hill, NSW, AU) and water was deionised (~18 megaohms) with a Millipore MilliQ system (North Ryde, NSW, AU). Fresh saxiphilin stock was isolated from wild catch of the centipede, Ethmostigmus rubripes and rat brain synaptosomes were freshly prepared as previously reported in:Llewellyn LE, Doyle J and Negri AP (1998) A high-throughput, microtiter plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin. Anal. Biochem. 261 (1): 51-56.Tritiated saxitoxin ([³H]-STX) was purchased from Amersham Pharmacia Biotech (UK), and toxin standards for HPLC analysis of PSTs were kindly donated by Prof. Y Oshima of Tohoku University, Japan. TTX standards were purchased from Calbiochem (Kilsyth, Vic., AU), STX dihydrochloride (STXdiHCl) was purchased from the Institute for Marine Biosciences, National Research Council, Canada, and GC-toxin standards were prepared as reported in:Negri A, Stirling D, Quilliam M, Blackburn S, Bolch C, Burton I, Eaglesham G, Thomas K, Walter J and Willis R (2003) Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum. Chem. Res. Toxicol. 16 (8): 1029-1033.Homogenisation was conducted on ice using a PRO250 tissue disruptor fitted with a 10 mm toothed head. Sonication was conducted in a Soniclean 500T bath sonicator (Transtek Systems, Selby Biolab), resting on ice for 10 min between sonic treatments. Centrifugation was carried out at 10,000xg for 20 min at 4°C, in a Hermle Z323K refrigerated centrifuge. Dried extracts were reconstituted in 0.05 M acetic acid (1.0 ml) and passed through a 10,000 MWCO centrifugal ultra-filter (Microcon, Millipore).Radio-receptor assays for TTX and PSTs were performed as described in:Llewellyn LE, Doyle J and Negri AP (1998). A high-throughput, microtiter plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin. Anal. Biochem. 261 (1): 51-56.Llewellyn LE, Negri AP, Doyle J, Baker PD, Beltran EC and Neilan BA (2001) Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis. Environ. Sci. Technol. 35 (7): 1445-1451.LC-FLD was performed using a Waters 600 HPLC (Milford, MA, USA) coupled to a Linear LC-305 fluorescent detector (Activon), Waters 717plus auto-sampler and a PCX 5100 post-column reactor (Pickering, Mountain View, CA, USA).PSTs were eluted as described in:Negri A and Llewellyn L (1998) Comparative analyses by HPLC and the sodium channel and saxiphilin ³H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical north west Australia. Toxicon 36 (2): 283-298.Oshima Y (1995) Post-column derivatization HPLC methods for paralytic shellfish poisons, in: Hallegraeff GM, Anderson DM and Cembella AD (Eds.) Manual on Harmful Marine Microalgae, vol. 33. Intergovernmental Oceanographic Commission Manuals and Guides, Paris, France, UNESCO, pp. 81-94.GC-toxin analogues were analysed as reported in:Negri A, Stirling D, Quilliam M, Blackburn S, Bolch C, Burton I, Eaglesham G, Thomas K, Walter J and Willis R (2003) Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum. Chem. Res. Toxicol. 16 (8): 1029-1033.Fluorescent derivatives of all PSTs were monitored at an excitation wavelength of 330 nm and an emission wavelength of 390 nm.TTX derivatives were analysed using a 5 µm, 250 x 2.1 mm Alltech Alltima C-18 column, eluted isocratically with 10 mM heptane sulfonic acid, then oxidised post-column in 4 M NaOH and monitored at an excitation wavelength of 415 nm and an emission wavelength of 485 nm as described in:Yotsu M, Endo A and Yasumoto T (1989) An improved tetrodotoxin analyzer. Agric. Biol. Chem. 53: 893-895.Liquid chromatography-mass spectrometry (LC-MS) was performed using a Shimadzu LC-10ADVP liquid chromatography system and Gilson 233XL sampling injector (Gilson, Inc., Middleton, WI, USA) which were coupled to a PE-SCIEX API 2000 triple quadrupole mass spectrometer (PE-SCIEX, Thornhill, Ontario, CA). LC separation was achieved on a TSK-Gel Amide-80 column (5 µm, 250x4.6 mm i.d. TosoHass) maintained at 40°C with an isocratic solution of 2 mM ammonium formate, 3.6 mM formic acid in 70% acetonitrile: water at 0.2 ml/min and flow was introduced into the Turbo Ionspray interface with a 9:1 (waste:MS) split. Retention times of STX and TTX standards were compared to sample extracts. Further LC-MS/MS experiments were performed using an Agilent 1100 Series LC coupled to an Esquire 3000+ quadrupole ion-trap mass spectrometer (Bruker Daltonics, Billerica MA, USA) fitted with an electrospray ionisation interface (HV capillaryC4 kV, skimmer voltage 40 V).

Notes

Credit
Robertson, Alison A, Dr (Principal Investigator)

Credit
Llewellyn, Lyndon E, Dr (Custodian)