NeuroTorp, a Lateral Flow Test Based on Toxin-Receptor affinity for in-situ early detection of cyclic imine toxins

NeuroTorp, a Lateral Flow Test Based on Toxin-Receptor affinity for in-situ early detection of cyclic imine toxins Fanny Noirmain1, Julie Dano2, Nathalie Hue3, Jesús M. Gonzalez-Jartin4, Luis M. Botana4, Denis Servent1,5 Stéphanie Simon2 and Rómulo Aráoz*1,5 1Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SIMoS/ Laboratoire Toxines, récepteurs et canaux ioniques, 91191 Gif-sur- Yvette, France. 2Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI/ Laboratoire d'études et de recherches en immunoanalyse, 91191, Gif-Sur-Yvette, France. 3Université Paris Saclay, ICSN, CNRS, UPR2301, Av. de la Terrasse, 91191 Gif sur Yvette, France. 4Universidad Santiago de Compostela, Facultad de Veterinaria, Departamento de Farmacologia, Lugo 27002, Spain. 5CNRS, ERL9004, 91191 Gif sur Yvette. *Corresponding author: Rómulo ARÁOZ CEA-Saclay SIMoS, Bât. 152 [email protected] Tel. +33 1 69 08 84 89 ABSTRACT The emergent cyclic imine toxins produced by marine dinoflagellates are potent antagonists of nicotinic acetylcholine receptors. Harmful algal blooms dominated by toxic dinoflagellates producing cyclic imine toxins have prompted in the past the closure of shellfish farms worldwide. Shellfish accumulate cyclic imine toxins following filter-feeding on toxic dinoflagellates vectoring them to humans. Herein is presented a lateral flow test for the detection of cyclic imine toxins based on three new concepts for test strips: i) the immobilization of lipoprotein vesicles in the test-line, ii) the high affinity of neurotoxins for their receptor targets and iii) the use of high porosity glass fiber filter membranes as support for the fabrication of the lateral flow test NeuroTorp (WO2017108115)1. Since nicotinic acetylcholine receptors represent ~30% of the total protein of the electric organ from Torpedo marmorata, the purified electrocyte membrane vesicles were used as a source of receptor and immobilized on the test-line. Biotin-α-bungarotoxin was used as toxin tracer for the LFT binding assay given its high affinity for nicotinic acetylcholine receptors. Finally, it is the first time that GF/C glass fiber membranes are used as support for the LFT NeuroTorp that ensures both, the immobilization of a complex lipoprotein sample and the capillary migration of the mobile phase. In absence of a nicotinic toxin, Biotin-α-bungarotoxin binds the nicotinic acetylcholine receptor and the use of neutravidin nanogold particle conjugate enables its visual detection. When nicotinic toxins are in the sample, they will enter in competition with the toxin tracer for binding the receptor. The inhibition degree is proportional to the concentration of the toxins in the sample. NeuroTorp is a ready-to-use low-cost point-of-care warning device for rapid and early detection of nicotinic neurotoxins in freshwater and marine environments by end-users. 1WO2017108115. Device for detecting neurotoxins and process for manufacture thereof. Aráoz R. et al. (2017). Keywords: Lateral flow test, nicotinic acetylcholine receptors, receptor ligand binding assay, cyclic imine toxins, phycotoxins detection, harmful algal blooms. 1. Introduction Marine phytoplankton plays a major role on biomass and O2 production and CO2 reduction at a global scale. Phytoplankton’s growth is characterized by episodic blooming covering large ocean surfaces at very high densities affecting the food-web dynamics and the eco-climatic parameters within aquatic environments [1]. The resulting harmful algal blooms (HABs) when dominated by toxin-producer species may be detrimental for marine wildlife, economical activities and Public Health. Shellfish that filter feed on toxic phytoplankton are primary vectors for the transfer of toxins to humans, provoking a series of health syndromes, some of them with fatal outcomes [2]. As a consequence, some marine toxins like saxitoxin, domoic acid, okadaic acid, pectenotoxin, yessotoxin and azaspiracid are regulated. Toxin monitoring applies to phytoplankton producers and shellfish, fish and seafood that accumulate and vectorize harmful toxins to humans [3]. As a consequence of global warming, the frequency of HABs events is increasing worldwide [4]. Cyclic imine toxins, ciguatoxins, tetrodotoxins and palytoxins are considered as emergent toxins in Europe [5]. Cyclic imine toxins (CiTXs) are fast acting neurotoxic compounds that kill mice by respiratory arrest within minutes after intraperitoneal injection or by gavage at lethal toxin doses [6, 7]. Of dinoflagellate origin, CiTXs move up fast the food chain through shellfish and may represent a potential threat for public health. The CiTX family (44 congeners) include gymnodimines, spirolides, pinnatoxins, pteriatoxins, prorocentrolides, spiroprorocentrolides and portimines that harbor a cyclic imine pharmacophore group in common [6, 8]. They are potent competitive antagonists of muscle and neuronal nicotinic acetylcholine receptors (nAChR) with affinities in the nanomolar range [9, 10]. Muscle nAChRs mediate fast neurotransmission at the neuromuscular junction level enabling muscle contraction and escape from predation, and play a crucial role at the autonomous nervous system for respiration control. Even if CiTXs are worldwide distributed and are often reported in shellfish for human consumption, they are not internationally regulated since no human fatalities are associated with this family of neurotoxins [3]. Recently however, the French agency for food safety recommended the inclusion of pinnatoxins in the list of marine regulated biotoxins establishing a guidance level for PnTX-G content in shellfish of 23 µg kg-1 [11]. Certain CiTXs congeners like 13-desmethyl spirolide-C (13-SPX-C), gymnodimine-A (GYM-A) and pinnatoxin-G (PnTX-G) can cross the brain-blood and the placental barriers [12-14]. Further, human subpopulations suffering from neuromuscular disorders like myasthenia gravis or Lambert- Eaton myasthenic syndrome can be vulnerable to CiTXs. Several liquid chromatography mass spectrometry methods and bioassays were developed for the detection of this class of emergent neurotoxins in Europe [15-18], however their use is restricted to reference laboratories for marine biotoxins. Lateral flow tests (LFT) are ready-to-use, low-cost, point-of-care diagnostic tools that are based on the use of antibodies, enzymes, nucleic acid probes or aptameres for auto-diagnose of pregnancy, blood glucose, infectious diseases, and for the detection of food contaminants, drugs, heavy metals and toxins [19-21]. In the present work, we report the development of a LFT to detect fast-acting neurotoxins based on three new approaches for this technology, that is: i) the high affinity of neurotoxins for their receptor targets, ii) the use of glass microfiber GF/C filter as the stationary phase for LFT and iii) the immobilization of complex lipoprotein membranes in the test-line. 2. MATERIAL AND METHODS 2.1 Reagents Cellulose fiber sample pads, laminated cards 60 mm × 301 mm and Estapor® microspheres were from MerkMillipore (Darmstadt, Germany). Whatman® GF/C glass mirofiber filters, BSA, sodium phosphate buffer, NHS-LC-Biotin, HAuCl4, trisodium citrate, were bought from Sigma-Aldrich (St Louis, MO, USA). Streptavidin Gold conjugated 40 nm was purchased from abcam (Cambridge UK). Monoclonal antibody RAB37, neutravidin, αbungarotoxin (α-BgTX), Biotin-α-BgTX conjugate were purchased from Thermo Fisher Scientific (Walthman, MA, USA). GYM-A and 13-SPX-C were purchased from NRC (Institute for Marine Biosciences, Halifax, NS, Canada). 13,19-didesmethyl spirolide-C (13,19-SPX-C) and 20-methyl spirolide-G (20-met-SPX-G) were bought from CIFGA (Lugo, Spain). Pinnatoxin-A (PnTX-A) and PnTX-G were synthesized in the Laboratory of Prof. Dr. A. Zakarian (Dep. of Chemistry and Biochemistry, University of California, Santa Barbara, CA, USA). 2.2 Preparation of Torpedo-electrocyte membranes vesicles for the test-line Torpedo marmorata electrocyte membrane vesicles were prepared as previously described [7]. The membranes were conserved at -80°C in 5 mM glycine until use. 2.3 Preparation of biotinylated RAB37 monoclonal antibody-latex bead microspheres conjugate for the control-line A volume of 100 µL of monoclonal antibody RAB37 (1 mg mL-1) prepared in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS) were mixed with 400 µL of 100 mM borate buffer, pH 9.5. After mixing, 6 µL of NHS-LC-Biotin (1 mg mL-1 in N,N-dimethyl formamide) was added to the antibody solution. The whole was incubated for 30 min at room temperature in the dark. Thereafter, 100 µL of 1M Tris buffer, pH 8 was added to the reaction mix and it was further incubated for 15 min as previously described. The protein concentration was calculated from the OD of the solution at 280 nm. A volume of 200 µL of biotinylated RAB37 antibodies was mixed with 200 µL of 1% polystyrene Estapor® microspheres prepared in PBS. The whole was incubated overnight in a rotatory shaker at 4°C. Free biotinylated RAB 37 antibodies were discarded by centrifugation (20 000g for 30 min at 4°C). The pelleted microbead-immobilized biotinylated RAB 37 antibodies were resuspended in 400 µL PBS buffer. The protein concentration was calculated from the difference between the OD at 280 nm of the biotinylated RAB37 solution and the OD at 280 nm of the supernatant. 2.4 Preparation of the colloidal neutravidin nanogold particle (NGP) conjugate The nanogold particles were synthesized by trisodium citrate reduction of HAuCl4. The size of the gold nanoparticles was controlled by dynamic light scattering. For the labelling of neutravidin with nanogold particles, 100 µL of neutravidin (1 mg mL-1) in 50 mM PBS, pH 7.5 was added to 1 mL colloidal gold. The whole was incubated at room temperature in a rotating shaker for 1 h in the dark. Then, 100 µL of 50 mM PBS, 0.1% BSA, pH 7.5 was added and the mixture was centrifuged at 14 000 rpm for 40 min. The supernatant was discarded and the pellet was suspended in 1 mL of 50 mM PBS, 0.1% BSA, pH 7.5. The mix was sonicated, vortexed and centrifuged as described. The neutravidin-NGP conjugate was resuspended in 250 µL of 50 mM PBS, 0.1% BSA, pH 7.5, aliquoted and stored at 4°C until use. 2.5 Construction of the LFT NeuroTorp A GF/C microfiber membrane strip of 30 cm long × 2.5 cm height was soaked in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) for 10 min. The GF/C filter was dried for an hour at 37°C in a forced air oven and was placed over a polystyrene sticking backing card. To dispense the nicotinic receptors onto the GF/C microfiber membrane, 500 µL of Torpedo-electrocyte membrane (500 µg mL-1 total protein) in TBS was filled in the test-line container of the Benchtop Dispenser Workstation XYX3050 (BioDot, UK). Similarly, 500 µL of RAB37-Biotin conjugated with latex bead microspheres (100 µg mL-1 protein) was filled in the control-line container. Torpedo-nAChR and RAB37-Biotin conjugated antibody were dispensed onto the GF/C membrane at a flowrate of 1 µL cm-1. After the membrane was dried as described, it was soaked in TBS, 0.1% BSA, pH 7.5 for 30 min at room temperature. Afterwards, the membrane was washed twice with water and then soaked in the preserving buffer TBS, 0.1% Tween, 7.5% glucose, pH 7.4. The membrane was dried for 1 h at 37°C in an air forced oven. Thereafter, the sample and absorbent pads strips were placed at both sides of the GF/C filter on the sticking backing card (Scheme 1). Then, 5 mm-wide strips were cut with a BioDot CM4000 Batch Cutting System. The membranes were stored in sealed bags at 4°C until use. 2.6 Toxin extraction from shellfish Two shellfish homogenates and four canned cockles were provided by CIFGA (Spain). Ten grams of canned cockles were homogenized using a waring blender and processed as described in [7, 17] according to Table 1. In parallel, 10 ml of canned cockle supernatant was mixed with an equal volume of acetone and CiTXs extraction was performed as described [17]. Briefly, the shellfish homogenates and the supernatant were mixed with acetone (see Table 1 for volumes used), vortexed three-times for one min and Roller vortexed for 15 min at room temperature in the dark. The suspensions were centrifuged for 15 min at 4 000 rpm at 20°C. The pellets were re-extracted with acetone as described, the supernatants were pooled and evaporated under a stream of azote at 40°C. The dried material was resuspended in methanol (Table 1) and conserved ay -20°C until use. 2.7 NeuroTorp protocol and data analysis Fifty µL of TBS-BSA, or TBS-BSA containing toxin sample were applied onto the sample pad. Once the whole was entirely migrated, 50 µL of the toxin tracer Biotin-α-BgTX (80 nM) was dispensed onto the sample pad. Following complete migration of the toxin tracer, 100 µL of neutravidin-NGP, extemporarily diluted 10-times with EIA-buffer (100 mM potassium phosphate, 150 mM NaCl, 0.1% BSA, 0.01% NaN3, 0.5% Tween 20, pH7.4), was added onto the sample pad. To improve the contrast 100 µL EIA-buffer was added to the sample pad. The results were visually evaluated after 15 min. Since NeuroTorp is a competitive inhibition test, the presence of a nicotinic toxin will displace the toxin-tracer in a concentration dependent manner: At 100% inhibition, the test control-line is colorless. In absence of inhibition, the test control line is red with a similar intensity as the control-line. For quantitation purposes, the NeuroTorp LFT strips were scanned with an LFT-reader (NgBiotech, Brest, France). The inhibition percent was calculated according to: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 100 × [(𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐿𝐹𝑇 𝑠𝑖𝑔𝑛𝑎𝑙 − 𝑆𝑎𝑚𝑝𝑙𝑒 𝐿𝐹𝑇 𝑠𝑖𝑔𝑛𝑎𝑙)/(𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝐿𝐹𝑇 𝑠𝑖𝑔𝑛𝑎𝑙)] 2.9 UPLC-MS/MS analysis of the sample extracts UPLC-MS/MS analysis of shellfish samples in the multi reaction mode was performed as described [17]. For quantitation purposes, calibration curves for GYM-A, 13,19-SPX-C, PnTX- A, 13-SPX-C, 20-met-SPX-G and PnTX-G in the range of 100 pM to 1 µM were built. The UPLC-chromatographic protocol was designed for the simultaneous detection and quantification of six cyclic imine toxin standards in a 10-min run. 2.10 Scanning Electron Microscopy (SEM) analysis of the LFT NeuroTorp NeuroTorp test-line strips were mounted on aluminium stubs and coated by sputtering with 3 nm of chromium (ACE600, Leica microsystems). The GF/C glass microfiber membranes were observed with a high-resolution Field-Emission SEM (GeminiSEM500, Zeiss) operating at 3 kV with a 15 or 20 µm objective aperture diameter. Secondary (topography) and backscattered (chemical contrast) electrons were collected in parallel with an in-lens detector and with an backscattered electron detector, respectively. Scan speed and line integration were adjusted during observation to optimize signal detection in both channels. 3. RESULTS AND DISCUSSION 3.1 Choice of an LFT membrane for immobilizing Torpedo-electrocyte lipoprotein vesicles NeuroTorp could be applied to the detection of competitive antagonists of nAChRs like the peptide neurotoxin α-BgTX (Figs. 3B-H) or the CiTX alkaloids (Figs. 4 and 5), or the freshwater cyanobacterial agonist anatoxin-a (data not shown). The degree of the inhibition is dependent on the potency of the neurotoxin tested. 3.2.4. CiTXs detection in shellfish extracts by NeuroTorp and by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Two shellfish homogenates and 4 cockle cans provided by CIFGA (Spain) were used to test NeuroTorp strips on real samples (Table 2). They were tested at 4 different dilutions containing 10, 5, 2.5 and 1.25% methanol, respectively. All the samples including the canned cockle (meat and supernatant) were positive for CiTXs, but, to a different extent (Fig. 5A). The test and control NeuroTorp strips illustrate that the inhibition binding was dose dependent. The Figs. 5B-E illustrate that methanol neither interferes with CiTXs detection nor with neutravidin- NGP binding to the biotinylated RAB37 antibody at the tested concentrations. All the shellfish samples were analyzed by UPLC-MS/MS for the simultaneous detection and quantification of GYM-A, 13,19-SPX-C, PnTX-A, 13-SPX-C, 20-me-SPX-G and PnTX-G. MS/MS fragmentation was optimized to determine the MRM transitions for each of the CiTX standards (Table 3). Linear calibration curves in the working range with an r2 ≥ 0.999 were obtained for each CiTX standard. The limits of detection (LOD) and limits of quantification (LOQ) were comparable with the reported literature [7, 15-17] (Table 3). ALTX- 01 was contaminated with 0.3 µg kg-1 GYM and 0.72 µg kg-1 13-SPX-C and with traces of PnTX-A. ALTX-02 contained 3.2 µg kg-1 GYM-A and 72 µg kg-1 13-SPX-C. Concerning the canned cockles for human consumption, both, the supernatant and flesh were contaminated with 1.2 and 4.7 µg kg-1 13-SPX-C, respectively. Traces of PnTX-A were detected in both samples, while traces of GYM-A could also be detected in cockle’s flesh samples (Table 3, Figs. 6A-D). Since CiTX are not regulated, it is usual to detect several members of this neurotoxin family in shellfish for human consumption [7, 15, 17]. PnTX-A is stable to thermal treatment [37]. It was seen that after heating at 100°C for 24 h only 20% of the PnTX-A underwent hydrolysis. The present results indicate that 13-SPX-C can resist the sterilization process to which canned cockles are submitted prior to commercialization. As the initial amount of 13-SPX-C are not known, the stability of this toxin cannot be calculated. This is the first report in which the presence of CiTXs is reported for canned shellfish. Conclusions Global Climate Change, water eutrophication and maritime activities have increased the frequency of HABs occurrence and the spreading of toxic species worldwide. The emergent CiTXs are often found in shellfish intended for human consumption since they are not regulated. Of concern is the high affinity of CiTXs for muscle and neuronal nAChRs subtypes given that they can cross the intestinal, brain blood and placental barriers. Current methods for detecting CiTXs for the shellfish industry include LC-MS and receptor-binding assay. These methods are performed in laboratories implying a time-gap between shellfish/ phytoplankton sampling and results delivery. The recently proposed guidelines for PnTX-G highlights the need to dispose of a simple and inexpensive method for detecting CiTXs. NeuroTorp provides the means for early, rapid and quantitative detection of nicotinic toxins in the field by end-users. NeuroTorp that is based on the affinity of CiTXs for the nAChRs introduces two innovative aspects for LFT technology: i) the use of a complex lipoprotein mixture as a source of receptors containing in addition ~400 different proteins (Torpedo marmorata-electrocyte membrane vesicles) instead of a single protein target immobilized in the test-line, and ii) the use of glass microfiber membranes as the stationary phase for LFT to immobilize lipoprotein vesicles. Torpedo-electrocyte membranes is a validated source of nAChRs of muscle-type. The use of a solely Torpedo marmorata electric fish as donor of electrocyte membranes provides enough material to manufacture hundred thousand of LFT NeuroTorp units. Thus, the number of animals sacrificed for toxicity screening in shellfish samples is highly reduced. It is the first time that glass microfiber filters of GF/C type are used as support for LFT applications. Herein is demonstrated that it is not necessary to functionalize the GF/C microfiber membrane to immobilize Torpedo-nAChR on the test-line. The spotted Torpedo-electrocyte vesicles anchor to neighboring glass microfibers forming lamellar microfilms that do not collapse following drying. These structures are stable through the time enabling specific interaction between the nAChR and competitive nicotinic ligands. The use of GF/C glass microfiber as the membrane support for NeuroTorp opens the way for immobilizing multiprotein complexes and even cellular membranes on the test-line of LFT. We have developed a fast and cost-effective early warning LFT device for field detection of cyclic imine toxins by end-users directly in the field. Acknowledgements The authors acknowledge the funding support from the LABEX LERMIT (DETECTNEUROTOX project, CDE 2017–001173 – RD 91 to RA), the NRBC-E Program (MULTITOX project, Fiche N° H35 to RA) and the INTERREG Atlantic Area (ALERTOXNET EAPA_317/2016 project to LMB and DS). The GDR PHYCOTOX is acknowledged for financial support to RA. We acknowledge Michaël Trichet from the IBPS electron microscopy core facility (https://www.ibps.sorbonne-universite.fr/en/core-facilities/imaging/electronmicroscopy-facility) and the support from the Région Île-de-France, from Sorbonne-Université and from CNRS. Table 1 EC50 of Biotin-α-BgTX to Torpedo-nAChR under different receptor concentrations. Torpedo-electrocyte membranes* (ng) Torpedo-nAChR** (ng) EC50 (nM) 1400 560 71.9 700 280 20.1 500 200 18.2 300 120 93.4 100 40 21.7 * Total protein concentration; ** Calculated concentration of nAChR considering that the nicotinic receptors represent ~40% of the total protein concentration of Torpedo-electrocyte membranes. Table 2 Shellfish samples: origin, processing and analyzed volumes. Sample Origin Sample processing Masse (g) ACN (mL)* MeOH (mL) to LFT (µL) to UPLC (µL) Mussels ALTX 01 CIFGA, Spain 5 20 2 1.25 – 10 5 Mussels ALTX 02 CIFGA, Spain 2.5 10 1 1.25 – 10 5 Cockles (can) SS CIFGA, Spain 10 40 4 1.25 – 10 5 Cockles (can) Flesh CIFGA, Spain 10 mL 40 4 1.25 – 10 5 * Shellfish samples were extracted twice with acetonitrile. Abbreviations: ACN: acetonitrile, MeOH: methanol, LFT: lateral flow test, UPLC: ultra-performance liquid chromatography. Table 3 UPLC-MS/MS conditions for CiTXs quantification. GYM-A 13,19-SPX-C PnTX-A 13-SPX-C 20-me-SPX-G PnTX-G MRM trans. (m/z) 508.4 > 490.4 678.4 > 164.1 712.4 > 164.0 692.4 > 444.4 706.5>688.4 694.4>676.4 Cone voltage (V) 50 50 65 60 65 75 Coll. energy (eV) 32 40 48 35 30 30 Ret. time (min) 4.83 6.19 6.23 7.31 8.54 8.99 LOD 10 pM or 0.010 pg/col. 100 pM or 0.14 pg/ col. 1 pM or 0.0014 pg/col. 100 pM or 0.14 pg/ col. 100 pM or 0.14 pg/col. 1 pM or 0.0014 pg/col. LOQ 1 nM or 1.0 pg/col. 1 nM or 1.35 pg/col. 10 pM or 0.014 pg/col. 1 nM or 1.38 pg/col. 1 nM or 1.41 pg/col. 10 pM or 0.014 pg/col. R 2 0.9999 0.9999 1.0000 0.9999 1.0000 0.9998 Abbreviations: UPLC-MS/MS: ultra-performance liquid chromatography-tandem mass spectrometry, GYM-A: gymnodimine-A, 13,19-SPX-C: 13,19-didesmethyl spirolide-C, PnTX- A: pinnatoxin-A, 13-SPX-C: 13-desmethyl spirolide-C, 20-me-SPX-G: 20-methyl spirolide-G, PnTX-G: pinnatoxin-G. MRM trans.: multiple reaction-monitoring mode transitions, Coll. Energy: collision energy, Ret. Time: retention time, LOD: limit of detection, LOQ: limit of quantification. Table 4 CiTX content in µg kg-1 shellfish determined by UPC-MS/MS Sample GYM-A (µg kg-1) 13,19-SPX-C (µg kg-1) PnTX-A (µg kg-1) 13-SPX-C (µg kg-1) 20-me-SPX-G (µg kg-1) PnTX-G (µg kg-1) Inhib. % (5% MeOH) Mussels ALTX 01 0.32 n.d. Traces 0.72 n.d. n.d. 41.9 ± 11 Mussels ALTX 02 3.2 n.d. n.d. 72 n.d. n.d. 76.4 ± 3.9 Cockles (can) SS n.d. n.d. Traces 1.2 n.d. n.d. 7.8 ± 5.7 Cockles (can) Flesh Traces n.d. Traces 4.7 n.d n.d. 41.6 ± 4.8 Abbreviations: GYM-A: gymnodimine-A, 13,19-SPX-C: 13,19-didesmethyl spirolide-C, PnTX-A: pinnatoxin-A, 13-SPX-C: 13-desmethyl spirolide-C, 20-me-SPX-G: 20-methyl spirolide-G, PnTX-G: pinnatoxin-G. References 1. Anderson, D. M.; Cembella, A. D.; Hallegraeff, G. M., Progress in Understanding Harmful Algal Blooms: Paradigm Shifts and New Technologies for Research, Monitoring, and Management. In Annual Review of Marine Science, Vol 4, Carlson, C. A.; Giovannoni, S. J., Eds. 2012; Vol. 4, pp 143-176. 2. Rodrigue, D. C.; Etzel, R. 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