First Identification of Palytoxin-Like Molecules in the Atlantic Coral

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First identification of PLTX-like molecules in the Atlantic coral species Palythoa canariensis María Fraga, Natalia Vilariño, M Carmen Louzao, Lucía Molina, Yanira López, Mark Poli, and Luis M. Botana Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01003 • Publication Date (Web): 24 Jun 2017 Downloaded from http://pubs.acs.org on July 4, 2017

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Analytical Chemistry

First identification of PLTX-like molecules in the Atlantic coral species Palythoa canariensis María Fraga1, Natalia Vilariño1*, M Carmen Louzao1, Lucía Molina2, Yanira López2, Mark Poli3 and Luis M Botana1* 1

Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de

Compostela, 27002 Lugo, Spain 2

Grupo de Investigación en Acuicultura (GIA) Instituto Canario de Ciencias Marinas P.O.

Box 56. 35200. Telde, Las Palmas, Canary Islands, Spain 3

Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases,

Fort Detrick, Maryland, USA *To whom correspondence should be addressed: Luis M. Botana, Natalia Vilariño Departamento de Farmacología Facultad de Veterinaria Campus Universitario 27002 Lugo Spain e-mail: [email protected], [email protected] Telephone and Fax: +34982822233

ACS Paragon Plus Environment

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ABSTRACT Palytoxin (PLTX) is a complex marine toxin produced by Zoanthids (Palyhtoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). Contact with PLTX-like compounds present in aerosols or marine organisms has been associated with adverse effects on humans. The worldwide distribution of producer species and seafood contaminated with PLTX-like molecules illustrates the global threat to human health. The identification of species capable of palytoxin production is critical for human safety. We studied the presence of PLTX analogues in Palythoa canariensis, a coral species collected in the Atlantic Ocean never described as a PLTX-producer before. Two methodologies were used for the detection of these toxins: a microsphere-based immunoassay that offered an estimation of the content of PLTX-like molecules in a Palythoa canariensis extract and an ultra-high pressure liquid chromatography coupled to ion trap with time of flight mass spectrometer (UPLC-IT-TOFMS) that allowed the characterization of the toxin profile. The results demonstrated the presence of PLTX, hydroxy-PLTX and, at least, two additional compounds with PLTX-like profile in the Palythoa canariensis sample. The PLTX content was estimated in 0.27 mg/g of lyophilized coral using UPLC-IT-TOF-MS. Therefore, this work demonstrates that Palythoa canariensis produces a mixture of PLTX-like molecules. This is of special relevance to safeguard human health considering Palythoa species are commonly used for decoration by aquarium hobbyists.


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INTRODUCTION Palytoxin (PLTX, Figure S-1) was described in 1971 through the identification of toxic organisms at Hana, Maui. The toxic specimens were described as zoanthids of the genus Palythoa1 and named Palythoa toxica2. Since then, the presence of PLTX-like molecules has been detected in zoanthids of the genera Palythoa, Zoanthus or Parazoanthus3,4. Most of them were collected from the Pacific Ocean1,5-9, but the presence of PLTX-like activity in Caribbean corals has been also described, although molecular identification of PLTX or its analogues was not provided10. Several PLTX-like compounds have been reported in these zoanthids: PLTX, homopalytoxin, bishomopalytoxin, neopalytoxin, deoxypalytoxin, and/or 42-hydroxy-palytoxin (42-OH-PLTX)5,11,12. Additional PLTX analogues have been found in dinoflagellates of the genus Ostreopsis2,13-17. Trichodesmium spp. cyanobacteria were also reported to synthesize PLTX and 42-OH-PLTX18. Initially, PLTX-like molecules were associated to Indo-Pacific waters, however, their repeated detection during Ostreopsis blooms in European coasts points to their geographical expansion13,19,20. The global distribution of PLTX-like compounds, their bioaccumulation in seafood21,22 and the reported poisonings, owed to direct contact with contaminated organisms or presence in marine or aquarium aerosols3,23,24, indicate these toxins are a sanitary threat. In fact, dermal, ocular and respiratory affections due to direct contact with coral or aerosol exposure during aquarium cleaning or dinoflagellate blooms are frequently reported5,23,25-27. In spite of a lack of regulation, diverse methods have been developed for the detection of PLTX-like molecules, including bioassays, chemical and biomolecular techniques4,28,29. Among them, analytical methods allowed the elucidation of PLTX structure and identification of other PLTX-like compounds8,28. Additionally, several immunoassays were developed using monoclonal and polyclonal anti-PLTX antibodies30-32 and used as screening tools for rapidly detecting presence/absence of PLTX analogues in samples.


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Although the presence of Palythoa species has been described along European coasts, they have never been reported as PLTX producers33-35. According to the Canary Island Catalog of Protected Species, the presence of two Palythoa species has been described in Canary Islands, Palythoa canariensis (Figure 1) and Palyhoa caribeorum36, being Palythoa canariensis more abundant37 . Actually, while Palythoa canariensis has been described in all Canary Islands except for Lanzarote38, Palythoa caribeorum presence has not been reported in Gran Canaria and La Gomera38. The aim of this work was to determine if the coral Palythoa canariensis produces PLTX-like molecules and to explore its toxins profile. For that purpose, we used an immunoassay and ultra-high pressure liquid chromatography coupled to ion trap–time of flight–mass spectrometry (UPLC-IT-TOF-MS). METHODS Materials PLTX (from Palythoa tuberculosa) was from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Palythoa tuberculosa extract was provided by Dr. Mark Poli (Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA). N-hydroxysuccinimide (NHS), sodium tetraborate decahydrate, jeffamine (2,2′-(ethylenedioxy)bis(ethylamine)),

boric

acid,

sodium

phosphate

monobasic,

ethanolamine and Tween-20 were from Sigma-Aldrich (Madrid, Spain). 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Pierce (Rockford, IL). Mouse monoclonal anti-PLTX antibody (anti-PLTX-mAb) was obtained as previously described39. Phycoerythrin Goat Anti-Mouse Ig (PE-Ab) was from Invitrogen (Eugene, OR). Sodium azide was from Fluka (Steinheim, Germany). Carboxylated microspheres (LC10019-01) were from Luminex Corporation(Austin, TX). Luminex sheath fluid, multiscreen 96 well filter plates, ACQUITY UPLC HSS T3 column (2.1 i.d.x100 mm, 1.8 µm particle size, 100 Å pore size), 33 mm Millex, Ultrafree-MC or -CL centrifugal filters


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(0.22 or 0.45 µm pore size)were purchased from Millipore (Billerica, MA). Formaldehyde (37%), sodium acetate anhydrous, di-sodium hydrogen phosphate anhydrous and sodium chloride were from reagent grade commercial sources. Acetonitrile was from UPLC grade, methanol from HPLC, formic acid from American Chemical Society, ethanol from PRS Panreac and 2-propanol was chemically pure. High purity water was obtained from a Millipore Direct 8/16 water system (Billerica, MA). Phosphate-buffered saline solution (PBS) was 130 mM NaCl, 1 mM NaH2PO4, 8.5 mM Na2HPO4, pH 7.4. PBS-BT solution was PBS with 0.1% w/v BSA and 0.01% v/v Tween-20. Buffer solutions were filtered through a 0.22 µm pore size filter before use.

Collection of Palythoa canariensis specimens Palythoa canariensis specimens (Figure 1) were identified37 and collected from La Laja Beach, Gran Canaria Island, Spain (latitude: 28.06072, longitude: -15.41997), where the presence of this coral species had been previously reported38. The corals were detached from the substrate during low tide using a sharp knife and kept in seawater while they were transported to the Canary Institute of Marine Sciences. In the laboratory they were washed, carefully brushed and lyophilized. These corals were collected under permission (503/2013) of Viceconsejería de Medio Ambiente y Ordenación Territorial (Regional Government of Canary Islands).

Extraction method for Palythoa canariensis samples An aliquot of 0.4 g of lyophilized Palythoa specimens (representing 1 g of wet material, approximately) was homogenized in a Dounce tissue grinder. Specimens were homogenized with 1 mL of 80% ethanol, the solvent was decanted in a tube and this procedure was repeated thrice, using the last step to collect also the remaining tissue in the same tube. This


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mixture was sonicated in an ice bath using 6 cycles of 30 s and incubated 16 h at room temperature, protected from light. Then, the sonication step was repeated and the mixture was centrifuged at 3000 g, at 20˚C for 10 min. This first supernatant was transferred to a fresh tube. The pellet was re-extracted as explained above using 4 mL of 80% ethanol but without an overnight incubation step. Both supernatants were pulled together, evaporated and reconstituted with 10 mL of 50% methanol. Before detection, the sample was filtered.

Microsphere-based immuno-detection assay for PLTX The analysis of the Palythoa canariensis extract was performed with a microsphere-based inhibition immunoassay using a Luminex 200™ system. The attachment of commercial PLTX (Wako) to microspheres and the immunodetection method were performed as in Fraga et al.31. Briefly, after the activation of microsphere carboxyl groups using EDC/NHS, 10 µg of PLTX were added to 2x106 pre-activated microspheres and allowed to react for 24 h. Finally, unreacted groups were blocked with 1 M ethanolamine. Microspheres were washed and stored for further use in PBS with 0.01% sodium azide. The microsphere-based immunoassay method consisted of a competition assay in which PLTX attached to the microspheres competes with PLTX in solution (calibration or unknown samples) for binding to a mouse anti-PLTX-mAb. The anti-PLTX-mAb bound to the microspheres is detected with a PE-labeled anti-mouse antibody. For the immunoassay 60 µL of sample or PLTX calibration solution (0.01-100 nM) were incubated 1 h with 60 µL of anti-PLTX-mAb (200 ng/mL). Then, this mixture was transferred to a microtiter filter plate containing 2x103 prewashed PLTX-coated microspheres for an overnight incubation (22 ºC). Later, the microspheres were washed and PE-Ab (0.5 µg/mL) was added for 1 h and, after another washing cycle, the microspheres suspended in PBS-BT were analyzed by the Luminex system. Microspheres were classified and PE fluorescence was quantified with 635 nm and


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532 nm wavelength lasers, respectively. Acquisition volume was 75 µL and minimum bead count number was 100.

Detection of PLTX-like molecules by UPLC-IT-TOF-MS UPLC coupled to IT-TOF-MS with an electrospray ionization (ESI) interface (Shimadzu, Kyoto, Japan) operated in positive mode, was used for the identification of PLTX-like molecules and quantification of PLTX in Palythoa canariensis. This method was performed as explained in Fraga et al.31 with slight modifications. Briefly, UPLC separation was carried out with an HSS T3 column coupled to an in-line filter kit. Mobile phases A and B were water and acetonitrile, respectively, acidified with 30 mM formic acid. Flow rate was 0.4 mL/min and temperature was maintained at 35˚C. Gradient was as follows: 20-45% mobile phase B during 11 min, 45-100% over 1.5 min, 100% for 1 min, 100-20% over 3 min and 20% for 9 min. All MSn were operated with the following ESI source conditions: nebulizing (N2) gas flow, 1.5 L/min; curved desolvation-line and heat block temperatures, 200˚C; drying gas pressure, 100 kPa; and detector voltage, 1.65 kV. PLTXs were analyzed using MS1 and MS2 with ion accumulation times of 60 and 120 msec, respectively. MSn data were used for the identification of PLTX and 42-OH-PLTX. MS1 spectra, with tri-, bi-, and monocharged ions, and the fragmentation pattern in MS2 served for identification and confirmation of PLTX: m/z 906.81 ([M+H+Ca]3+ or [M+2H+K]3+) and 1359.71 ([M+Ca]2+ or [M+H+K]2+); and 42-OH-PLTX: m/z 912.15 ([M+H+Ca]3+ or [M+2H+K]3+) and 1367.72 ([M+Ca]2+ or [M+H+K]2+)28,31,40. IT-TOF-MS resolution at m/z 1000 is 10000 and it does not permit to distinguish between some pairs of ion adducts as Ca2+ and K+ or Na+ and Mg2+15. MS2 precursors and energy (E) and collision gas (Cg) were: m/z 906.81 with E 30% and Cg 25%, m/z 912.15 with E 60% and Cg 55%, m/z 864.78 and m/z 960.51 with E 55% and Cg 50%. PLTX calibration solutions were prepared in 10% methanol from a 403 ng/µL (150 µM)


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stock solution (Wako). Calibration levels range was 0.1-20 ng/µL. Injection volume was 5 µL. A sodium trifluoroacetate solution was used for mass calibration. LOD and LOQ for PLTX were 190 and 650 ng/mL, respectively. Data analysis All experiments were performed in duplicate. The immunoassay results are expressed as mean±SEM of three independent experiments. The results obtained with calibration solutions were fitted using GraphPad Prism 5.0 to a four-parameter logistic equation obtained with a nonlinear regression fitting procedure: Y=Rhi+(Rlo-Rhi)/(1+10^((LogIC50-X)*HillSlope)), where Rhi is the response at infinite concentration, Rlo is the response at 0 concentration, IC50 is the half maximal inhibitory concentration and X is the logarithm of concentration to base 10. Data obtained with IT-TOF-MS were analyzed using the LabSolutions-LCMS software (Shimadzu, Kyoto, Japan).

RESULTS AND DISCUSSION Chromatographic and mass spectrum profiles of Palythoa canariensis The Palythoa canariensis extract was analyzed by UPLC-IT-TOF-MS to explore its profile of PLTX-like molecules. A PLTX commercial standard (from Palythoa tuberculosa) and a Palythoa tuberculosa extract were also analyzed to compare MS spectra and retention times for toxin identification. The parameters used for the identification of PLTX-like molecules were: a) retention time (RT), b) MS1 spectra containing tri-, bi-, and monocharged ions with their respective adducts and water losses, c) presence of the A-moiety fragment (C8-C9, Figure S-1)28, and d) MS2 spectra of the most intense MS1 tricharged ion. In addition, theoretical m/z ion patterns for MS1 were established for tri-, bi-, and monocharged ions with their respective adducts (Ca2+ or K+, Na+ or Mg2+) and/or water losses to help identify molecules of the PLTX family.


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Palythoa canariensis contained different compounds that share chromatographic and spectral features with PLTX-like molecules. Four main compounds displayed intense tricharged ions of m/z 906.81, 912.15, 864.79 and 960.51, and will be named as molecules A, B, C and D respectively for description purposes (Figure 2A).

M/z 906.81 In the Palythoa canarienesis m/z 906.81 showed a main peak at RT 7.2 min (Figure 2A, molecule A). The study of the MS1 spectrum revealed the presence of tri-, bi- and monocharged ion clusters, with the highest intensity corresponding to m/z 906.81, 1105.61 and, 2282.26, respectively (Figure 3A). The main peak of PLTX commercial standard, also with a m/z 906.81 tricharged ion, eluted at a similar RT, 7.2 min (Figure 2B). Its MS1 spectrum contained clusters of tri-, bi- and monocharged ions coincident with those detected for molecule A in Palythoa canariensis (Figure 3A, Figure S-2A). Some of these ions are characteristic of PLTX, such as 906.81 ([M+2H+K]3+ or [M+H+Ca]3+), 1359.72 ([M+H+K]2+ or [M+Ca]2+) or 2282.25 ([M+H-A moiety-4H2O]+) (Figure 3A, Figure S-2A). Additionally, among the monocharged ions, the ion corresponding to the whole molecule, 2680.50 ([M+H]+), was identified in Palythoa canariensis, along with several dehydration steps (Figure 3A.v) and B-moiety (Figure 3A.iv). PLTX B-moiety bicharged ions were also found in Palythoa canariensis (Figure 3A.ii-iii) and PLTX standard (Figure S-2A.ii-iii), matching those expected for PLTX (Table S-1). The A-moiety ion (m/z 327.19), product of some PLTX-like molecules fragmentation at C8-C9 (Figure S-1), as well as its dehydrated form (m/z 309.18)28, were observed in Palythoa canariensis and PLTX standard MS1s (Figure 3A.vi, Figure S-2A.iv). This fragment was also detected in the MS2 spectrum obtained after fragmentation of m/z 906.81, in Palythoa canariensis and PLTX standard (Figure 3B.i, Figure S-2B.i). The fragmentation pattern of m/z 906.81 from Palythoa canariensis was


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similar to the PLTX standard (Figure 3B.i-iii, Figure S-2B.i-iii). In both cases MS2 spectra displayed other characteristic PLTX fragments: m/z 452.24 (internal fragmentation between C8-9 and C48-49 with a dehydration step) and m/z 596.32 (external fragmentation at C4546), as well as other fragments reported for PLTX in a previously published work40 (Figure 3B.i, Figure S-2B.i, Table S-2). Therefore, molecule A of Palythoa canariensis with a main m/z 906.81, tricharged ion was identified as PLTX. In addition to this main PLTX peak, four adjacent minor peaks of m/z 906.81 appeared in Palythoa canariensis eluting at 6.06, 6.84, 7.05 and 7.61 min (Figure 2A). Similarly, PLTX standard from Wako showed two minor peaks of m/z 906.81 with RTs of 7.05 and 7.63 min (Figure 2B). The analysis of MS1 and MS2 (precursor ion: m/z 906.81) of these Palythoa canariensis molecules seemed to match the spectra obtained for the equivalent peaks in PLTX standard. Because these peaks from both samples had different RT than PLTX, to evaluate the similarity of these molecules to PLTX, the LabSolutionsLCMS software (Shimadzu, Kyoto, Japan) was used to generate a library with the compiled information about ionization (MS1) and fragmentation (MS2) patterns of PLTX standard. Then, this library was used to compare the minor molecules with PLTX. For the molecules with RTs 6.06 and 6.84 min in Palythoa canariensis, the software provided rates of similarity for MS1 spectral data of 50% and 86%, respectively (Table S-3). Due to the low peak intensity (Figure 2A) some ions were not present, which would reduce MS1 percentage of similarity and preclude the comparison of MS2 data with PLTX MS2. However, the molecules with RTs 7.05 and 7.62 min, in the coral sample and standard, showed fair similarity rates for MS1 and MS2 to PLTX (RT 7.2 min). When the spectra of these molecules in Palythoa canariensis were compared against MS1 and MS2 spectra of PLTX, the percentage of similarity was higher than 82% for MS1 and 67% for MS2 for both molecules (Table S-3). These high similarity rates with PLTX and their chromatographic RTs (Table S-3, Figure 2A and 2B) suggest both compounds may


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be PLTX stereoisomers. Co-existence of PLTX with some analogues has been previously described in Palythoa tuberculosa12,31,41, therefore it could occur in other coral species. In fact, the elution of PLTX stereoisomers at different RTs has been demonstrated in corals and dinoflagellates12,14,16,17. For example, isopalytoxin, a PLTX isomer found in Palythoa tuberculosa, was demonstrated to elute later than PLTX12, as the molecule at RT 7.63 min. Although further studies would be necessary to establish their chemical structure, those four minor compounds observed in Palythoa canariensis with m/z 906.81 might be stereoisomers co-existing with PLTX.

M/z 912.15 A compound with a main tricharged ion of m/z 912.15 eluting at RT 7.0 min was found in the same Palythoa canariensis sample (Figure 2A, compound B). In spite of its low intensity, triand bicharged ion clusters were clearly observable in MS1, with m/z 912.15 and 1321.73 being among the most intense ions, respectively (Figure 4A.i and 4A.iii). These data were compared with the 42-OH-PLTX spectra (Figure S-3A.i-v), the most abundant component of the Palythoa tuberculosa extract used to identify this molecule31. In this extract the main compound with m/z 912.15 eluted at 7.0 min (Figure 2C). Among the tri-, bi- and monocharged ions with higher intensity in the Palythoa tuberculosa extract for 42-OH-PLTX were m/z 912.15 ([M+2H+K]3+ or [M+H+Ca]3+), 1321.73 ([M+2H-3H2O]2+), and 2298.26 ([M+H-A moiety-4H2O]+), respectively (Figure S-3A.i-v). The RT and MS1 spectrum for 42OH-PLTX in Palythoa tuberculosa were almost identical to those found for molecule B from Palythoa canariensis (Figure 2A and 2C, Figure 4, Figure S-3, Table S-4). In addition, m/z 2697.45 ([M+H]+) ion, characteristic of 42-OH-PLTX was observed in undiluted Palythoa canariensis extract (Figure 4A.iv). Tri- and bicharged ions (MS1) were coincident with the theoretical ion pattern of 42-OH-PLTX (Figure 4A.i-iii, Figure S-3A.i-iii, Table S-4). The


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fragmentation of m/z 912.15 yielded an MS2 spectrum very similar for Palythoa canariensis and Palythoa tuberculosa (Figure 4B.i-iii, Figure S-3B.i-iii, Table S-5). In both cases m/z 912.15 MS2, displayed an up-shift of 16/z mass units for the fragments that contained C42 of the original molecule. For example, m/z 604.32 of 42-OH-PLTX MS2 (external cleavage at C45-46, Figure 4B.i, Figure S-3B.i, Table S-5) has an increase of 8 versus the homologous bi-charged fragment reported for PLTX (Figure S-2B.i, Table S-2 and40). Therefore, the spectra obtained in MS1 and MS2 confirmed the presence of an additional oxygen in the Bside of the molecule versus PLTX42, and allowed the identification of the main peak at m/z 912.15 in Palythoa canariensis as 42-OH-PLTX.The co-existence of 42-OH-PLTX and PLTX has been previously demonstrated in different Palythoa species11,31.

M/z 864.79 In Palythoa canariensis a low intensity m/z 864.79, tricharged ion eluting at RT 7.5 min was also found (Figure 2A, compound C). MS1 displayed the presence of tri- and bicharged ion clusters, being m/z 864.79 and 1241.68 the ions with higher intensity (Figure 5A.i and 5A.iii). In the PLTX commercial standard a compound with a predominant peak at m/z 864.79, also with low intensity, eluted at RT 7.4 min (Figure 2B), and displayed a similar MS1 ion pattern to the one in Palythoa canariensis (Figure S-4A). MS1 spectra of this molecule contained triand bicharged ions with several subsequent dehydrations and the potential presence of adducts. A theoretical ionization pattern was predicted for this molecule based on PLTX and 42-OH-PLTX ionization (Table S-6). The most intense tri- and bicharged ions of m/z 864.79 and 1241.68, could be assigned to [M+2H+K]3+ or [M+H+Ca]3+ and [M+2H-4H2O]2+, respectively (Figure 5A.i and 5A.iii, Figure S-4A.i and S-4A.iii, Table S-6). Monocharged ion 2554.38 ([M+H]+) was also found for this molecule in undiluted Palythoa canariensis extract (Figure 5A.iv). Additionally, bicharged ion clusters gathered around m/z 1105.61,


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present in the coral sample and standard (Figure 5A.ii, Figure S-4A.ii), matched those found for PLTX (Figure S-2A.ii), which seems to indicate this molecule and PLTX possess the same B-moiety (Figure S-1). A probable A-moiety ion was not detected for this molecule neither in Palythoa canariensis nor in the standard. However, the absence of the A-moiety ion might be due to different reasons: the low intensity of the signal, which probably indicates a low concentration, and the analytical method, which was optimized to minimize the fragmentation of PLTX-like compounds before their entrance in the IT31. In spite of a missing A-moiety ion, the coincidence of bicharged ions around m/z 1105.61 (Figure 5A.ii, Figure S-4A.ii) with those found in PLTX (Figure S-2A.ii) suggests the molecular difference between PLTX and compound C might reside in the A-moiety. Modifications of PLTX-like molecules in this region have been previously described for different compounds from corals or dinoflagellates8,14. An estimation of the A-moiety mass for compound C found in Palythoa canariensis, assuming a B-moiety equal to PLTX’s, yielded a m/z of 201.08. In Palythoa canariensis m/z 864.79 fragmentation produced several dehydration products of the tricharged ion, and some low intensity smaller fragments (Figure 5B) coincident with fragmentation of the same mass in the standard (Figure S-4B). These fragments could be related to some previously reported internal fragments of PLTX40, such as m/z 394.22, corresponding to cleavage between C8-9 and C43-44 (Figure 5B.i, Figure S-4B.i, Figure S2B.i, Table S-2). Although some other fragments were observed in the standard (m/z 372.20 from cleavage between C8-9 and C41-42 or m/z 452.24 from cleavage between C8-9 and C48-49, in Figure S-4B.i), the information was not enough to clearly identify the molecule. This compound, with a predominant ion at m/z 864.78, has been reported as a minor contaminant of the commercial PLTX standard in a previous study41. Nevertheless, the presence of tri- and bicharged ions in MS1 and MS2 and their similarity with the theoretical pattern established for the ionization of PLTX-like compounds suggest this molecule belongs


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to the PLTX class, although it should be confirmed by further analysis when sufficient material is available.

M/z 960.51 Finally, a fourth compound with suspected PLTX-like mass spectrum, was present in Palythoa canariensis with a main tricharged ion of m/z 960.51 (compound D, Figure 2A). This molecule eluted before PLTX and 42-OH-PLTX, at RT 5.9 min (Figure 2A). MS1 spectrum presented clusters of bi- and tricharged ions, the higher intensities corresponding to m/z 1449.27 and m/z 960.51, respectively (Figure 6A.i-iii). This ion profile was compared to a theoretical ionization pattern predicted from previously reported MS1 spectra of PLTX-like molecules (Table S-7). Based on this ionization pattern, the doubly charged, m/z 1449.27 ion might correspond to [M+2H-H2O]2+ and the tricharged m/z 960.51 could match [M+3H2H2O]3+ (Figure 6A.i and 6A.iii, Table S-7), the other main cluster of bicharged ions, around m/z 1250.15 would be the dehydrated B-moiety (Figure 6A.ii, Table S-7). A-moiety ion, m/z 327.19, was absent in MS1 but appeared in MS2 after m/z 960.51 fragmentation (Figure 6B.i). Concurrently, the A-moiety dehydrated form ([A-moiety-H2O]+), serial dehydration steps of the bicharged B-moiety ion (around m/z 1250.17) and several dehydrations of the parent ion were observed in MS2 (Figure 6B.i and 6B.ii). The presence of the characteristic A-moiety fragment of PLTX molecule in MS2, and the ion pattern in MS1 suggest that compound D eluting at RT 5.9 min, could belong to the PLTX family. However, further studies with a higher amount of molecule would be necessary to disclose its chemical formula.

Quantification of PLTX-like compounds in Palythoa canariensis The Palythoa canariensis extract was analyzed by a microsphere-based immunoassay and UPLC-IT-TOF to obtain an estimate of the content of toxins belonging to the PLTX class.


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In the immunoassay, the Palythoa canariensis extract was assayed simultaneously with calibration solutions prepared in PBS-BT using PLTX from Wako as calibrant. IC50 of the calibration curve was 4.76±0.32 ng/mL, LOD, estimated as IC2043, was 1.75±0.45 ng/mL and IC80 was 19.0±1.6 ng/mL (Figure S-5). The extract analysis displayed concentrations of PLTX-like molecules higher than the upper value of the assay dynamic range. Subsequently, several dilutions of the coral extract, from 1:15000 to 1:750000 (v/v) in PBS-BT, were tested and provided results within the dynamic range of the calibration curve. The 1:15000 (v/v) extract dilution was assayed thrice and the averaged results were used for the final toxin content calculation. The concentration of PLTX-like molecules in the extract was estimated at 2.32±0.26 mg PLTX-like molecules/g lyophilized coral (Table S-8). For PLTX content estimation in Palythoa canariensis with UPLC-IT-TOF a 1:2 (v/v) extract dilution was used. PLTX (m/z 906.81, RT 7.2) was quantified using the commercial standard, yielding a concentration of 0.27 mg PLTX/g lyophilized coral (Table S-8). The concentration in the sample of the other PLTX-like compounds with a main m/z 906.81 ion (RT≠7.2) was also evaluated using PLTX as reference because there is no standard available for these molecules. The estimated concentration was 0.11 mg PLTX-like molecules/g lyophilized coral (Table S-8). The concentration of molecules B (42-OH-PLTX), C and D, represented by m/z 912.15, 864.79 and 960.51, was estimated using PLTX as calibrant, due to the lack of specific standards. Molecules B, C and D concentration was estimated at 0.07, 0.05, and 0.04 mg PLTX-like molecules/g lyophilized coral (Table S-8). The sum of the concentration of all PLTX-like compounds in the extract was 0.55 mg PLTX-like molecules/g lyophilized coral (Table S-8). The difference between the immunoassay (2.32 mg/g) and the analytical quantification (0.55 mg/g) can be due to diverse factors: a) cross-reactivity of the anti-PLTX antibody with the analogues present in the sample, b) high sensitivity of the immunoassay (LOD 1.75 ng/mL)


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versus UPLC-IT-TOF-MS (LOD 190 ng/mL), c) lack of certified standards for PLTX analogues, which precludes their correct quantification by analytical methods and d) semiquantitative nature of the immunoassay. Actually, the estimation of the PLTX-like molecules content by the immunoassay was made under the assumption that all analogues in the sample had the same affinity for this anti-PLTX antibody, which would be highly improbable. In fact, different antibody cross-reactivity has been suggested for PLTX and 42OH-PLTX in previous publications, with 42-OH-PLTX having a considerably lower affinity31,42. Unfortunately, the lack of certified reference material for these molecules precludes adequate cross-reactivity studies. A matrix interference effect in the immunoassay causing an overestimation of toxin content might also be responsible for the difference of toxin estimates by both methods, considering the high dilution of the Palythoa canariensis extract. However, in a previous study a high dilution of the Palythoa tuberculosa extract was also necessary to estimate its toxin content by the immunoassay in similar conditions, and no coral matrix effect was present31. Therefore, previous studies indicate that the immunoassay can be used in coral samples as a semiquantitative assay without matrix interference, although differences between coral species cannot be overruled. Unfortunately, no blank Palythoa canariensis specimens were available for a thorough analysis of the matrix effect. For the estimation of PLTX-like molecules content by UPLC-IT-TOF-MS some assumptions were made as well, such as different analogues having the same ionization efficiency as PLTX. Both methods have important limitations at the moment for accurate determination of toxin content owed to the lack of certified reference materials. In addition, the immunoassay would be only suitable as a semiquantitative, screening method for samples with complex toxin profiles (mixture of several analogues). These results demonstrate, for the first time, the presence of PLTX, 42-OH-PLTX and six minor PLTX-like molecules in Palythoa canariensis, a coral species from the Atlantic Ocean.


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The detection of toxins of the PLTX class has been previously described in dinoflagellates from the Atlantic13,44,45 but these molecules had not been identified in Atlantic zoanthids before. Although taxonomic classification of zoanthids is still controversial due to recent molecular analysis, the morphologic characteristics and specific location of the collection point support the identification of the specimens used in this study as Palythoa canariensis. This finding reflects a global distribution of PLTX producers and suggests the risk of human exposure to these toxins might have been underestimated. Actually, Palythoa species are commonly used for decoration by aquarium hobbyists and cases of PLTX-related toxicity have been reported associated to this use3,46. The identification of PLTX producers is also critical for adequate environmental impact evaluation or risk assessment of accidental exposure during recreational activities.

ACKNOWLEDGMENTS The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía,

Industria

y

Competitividad, AGL2014-58210-R, AGL2016-78728-R

(AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD and ITC-20161072. From the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA. SAFETY Palytoxin standard and contaminated material should be handled with care, gloves and eye protection should be worn at all times. Appropriate disposal methods should be utilized.

SUPPORTING INFORMATION


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PLTX structure. Mass spectra of PLTX and PLTX-like molecules in the PLTX standard and Palythoa

tuberculosa extract. Similarity of mass spectra for molecule identification.

Immnunoassay calibration curve. Toxin content of Palythoa canariensis extract.

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(14) Ciminiello, P.; Dell'Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Grauso, L.; Tartaglione, L.; Guerrini, F.; Pistocchi, R. Rapid Commun Mass Spectrom 2010, 24, 2735-2744. (15) Garcia-Altares, M.; Tartaglione, L.; Dell'Aversano, C.; Carnicer, O.; de la Iglesia, P.; Forino, M.; Diogene, J.; Ciminiello, P. Anal Bioanal Chem 2015, 407, 1191-1204. (16) Suzuki, T.; Watanabe, R.; Uchida, H.; Matsushima, R.; Nagai, H.; Yasumoto, T.; Yoshimatsu, T.; Sato, S.; Adachi, M. Harmful Algae 2012, 20, 81-91. (17) Uchida, H.; Taira, Y.; Yasumoto, T. Rapid Commun Mass Spectrom 2013, 27, 19992008. (18) Kerbrat, A. S.; Amzil, Z.; Pawlowiez, R.; Golubic, S.; Sibat, M.; Darius, H. T.; Chinain, M.; Laurent, D. Mar Drugs 2011, 9, 543-560. (19) David, H.; Laza-Martínez, A.; Orive, E.; Silva, A.; Moita, M. T.; Mateus, M.; De Pablo, H. Harmful algae news 2012, 45, e13. (20) Sechet, V.; Sibat, M.; Chomérat, N.; Nézan, E.; Grossel, H.; Lehebel-Peron, J.-B.; Jauffrais, T.; Ganzin, N.; Marco-Miralles, F.; Lemée, R.; Amzil, Z. Cryptogamie, Algologie 2012, 33, 89-98. (21) Aligizaki, K.; Katikou, P.; Milandri, A.; Diogène, J. Toxicon 2011, 57, 390-399. (22) Amzil, Z.; Sibat, M.; Chomerat, N.; Grossel, H.; Marco-Miralles, F.; Lemee, R.; Nezan, E.; Sechet, V. Mar Drugs 2012, 10, 477-496. (23) Deeds, J. R.; Schwartz, M. D. Toxicon 2010, 56, 150-162. (24) Tichadou, L.; Glaizal, M.; Armengaud, A.; Grossel, H.; Lemee, R.; Kantin, R.; Lasalle, J. L.; Drouet, G.; Rambaud, L.; Malfait, P.; de Haro, L. Clin Toxicol (Phila) 2010, 48, 839844. (25) Hoffmann, K.; Hermanns-Clausen, M.; Buhl, C.; Büchler, M. W.; Schemmer, P.; Mebs, D.; Kauferstein, S. Toxicon 2008, 51, 1535-1537.


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(26) Nordt, S. P.; Wu, J.; Zahller, S.; Clark, R. F.; Cantrell, F. L. J Emerg Med 2011, 40, 397399. (27) Rumore, M. M.; Houst, B. M. International Journal of Case Reports and Images (IJCRI) 2014, 5, 501-504. (28) Ciminiello, P.; Dell'Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Tartaglione, L. Toxicon 2011, 57, 376-389. (29) Louzao, M. C.; Fraga, M.; Vilariño, N. In Phycotoxins; John Wiley & Sons, Ltd, 2015, pp 113-135. (30) Campbell, K.; McNamee, S. E.; Huet, A. C.; Delahaut, P.; Vilarino, N.; Botana, L. M.; Poli, M.; Elliott, C. T. Anal Bioanal Chem 2014. (31) Fraga, M.; Vilarino, N.; Louzao, M. C.; Fernandez, D. A.; Poli, M.; Botana, L. M. Anal Chim Acta 2016, 903, 1-12. (32) Zamolo, V. A.; Valenti, G.; Venturelli, E.; Chaloin, O.; Marcaccio, M.; Boscolo, S.; Castagnola, V.; Sosa, S.; Berti, F.; Fontanive, G.; Poli, M.; Tubaro, A.; Bianco, A.; Paolucci, F.; Prato, M. ACS Nano 2012, 6, 7989-7997. (33) Calado, R. Sci Mar 2006, 70, 389-398. (34) Carreiro-Silva, M.; Braga-Henriques, A.; Sampaio, I.; de Matos, V.; Porteiro, F. M.; Ocana, O. Ices J Mar Sci 2011, 68, 408-415. (35) Williams, R. B. Ophelia 2000, 52, 193-206. (36) BOE-A-2010-9772. 2010, 150. (37) Espino-Rodríguez F., B.-L. A., Tuya-Cortés F., Haroun-Tabraue R.J. Guía visual de Especies Marinas de Canarias, 2nd ed.; Oceanográfica: Divulgación, Educación y Ciencia S.L.: Las Palmas de Gran Canaria, 2009, p 481. (38) Gobierno de Canarias, Consejería de Medio Ambiente y Ordenación Territorial, Dirección General del Medio Natural. Evaluación de especies amenazadas de Canarias:


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http://www.gobiernodecanarias.org/medioambiente/piac/temas/biodiversidad/medidas-yfactores/flora-fauna/conservacion-especies/conservacion-especies-amenazadas/evaluacion2009/interes-especial/, 2009. (39) Bignami, G. S.; Raybould, T. J.; Sachinvala, N. D.; Grothaus, P. G.; Simpson, S. B.; Lazo, C. B.; Byrnes, J. B.; Moore, R. E.; Vann, D. C. Toxicon 1992, 30, 687-700. (40) Ciminiello, P.; Dell'Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Grauso, L.; Tartaglione, L. J Am Soc Mass Spectrom 2012, 23, 952-963. (41) Ciminiello, P.; Dell'Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Tartaglione, L.; Benedettini, G.; Onorari, M.; Serena, F.; Battocchi, C.; Casabianca, S.; Penna, A. Environ Sci Technol 2014, 48, 3532-3540. (42) Ciminiello, P.; Dell'Aversano, C.; Dello Iacovo, E.; Forino, M.; Tartaglione, L.; Pelin, M.; Sosa, S.; Tubaro, A.; Chaloin, O.; Poli, M.; Bignami, G. J Nat Prod 2014, 77, 351-357. (43) Fraga, M.; Vilarino, N.; Louzao, M. C.; Campbell, K.; Elliott, C. T.; Kawatsu, K.; Vieytes, M. R.; Botana, L. M. Anal Chem 2012, 84, 4350-4356. (44) Nascimento, S. M.; Corrêa, E. V.; Menezes, M.; Varela, D.; Paredes, J.; Morris, S. Harmful Algae 2012, 13, 1-9. (45) Riobó, P.; Paz, B.; Franco, J. M. Analytica Chimica Acta 2006, 566, 217-223. (46) Tartaglione, L.; Pelin, M.; Morpurgo, M.; Dell'Aversano, C.; Montenegro, J.; Sacco, G.; Sosa, S.; Reimer, J. D.; Ciminiello, P.; Tubaro, A. Toxicon 2016, 121, 41-50.


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Legend to the figures Figure 1. Wild Palythoa canariensis specimens. A) Closed polyps at low tide. B) Opened polyps when submerged at high tide. Figure 2. Chromatograms of molecules with PLTX-like m/z profile in Palythoa canariensis, PLTX standard and Palythoa tuberculosa extract. A) Chromatographic separation of m/z 906.81 (RT 7.2), 912.15 (RT 7.0), 864.79 (RT 7.5) and 960.51 (RT 5.9) found in Palythoa canariensis. A, B, C and D indicate the main peaks of each m/z. B) Chromatographic separation of m/z 906.81, 912.15 and 864.79 in the PLTX commercial standard (Palythoa tuberculosa, Wako). C) Chromatographic separation of m/z 906.81 and 912.15 in the Palythoa tuberculosa extract. Figure 3. Ionization and fragmentation spectra of the molecule eluting at RT 7.2 min in Palythoa canariensis (molecule A, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bi-; iv and v) monocharged and vi) A-moiety ions. B) Segments of MS2 spectrum obtained from the fragmentation of the m/z 906.81 tricharged ion: i) m/z 300-650; ii) m/z 700-905 and iii) m/z 1050-1225. Figure 4. Ionization and fragmentation spectra of the molecule eluting at RT 7.0 min in Palythoa canariensis (molecule B, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bicharged; and iv) monocharged ions. B) Segments of MS2 spectrum obtained after fragmentation of the m/z 912.15 tricharged ion: i) m/z 300-660; ii) m/z 700-915 and iii) m/z 1125 to 1195. Figure 5. Ionization and fragmentation spectra of the molecule eluting at RT 7.5 min in Palythoa canariensis (molecule C, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bicharged; and iv) monocharged ions. B) Segments of MS2 spectrum obtained after fragmentation of the m/z 864.79 tricharged ion: i) m/z 300-670; ii) m/z 640-880 and iii) m/z 1000-1500.


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Figure 6. Ionization and fragmentation spectra of the molecule eluting at RT 5.9 min in Palythoa canariensis (molecule D, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bicharged ions. B) Segments of MS2 spectrum obtained from the fragmentation of the m/z 960.51 tricharged ion: i) m/z 300-660; ii) m/z 880-1000 and iii) m/z 1180-1280.


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For TOC only.


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Figure 1. Wild Palythoa canariensis specimens. A) Closed polyps at low tide. B) Opened polyps when submerged at high tide. 68x75mm (300 x 300 DPI)



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Figure 2. Chromatograms of molecules with PLTX-like m/z profile in Palythoa canariensis, PLTX standard and Palythoa tuberculosa extract. A) Chromatographic separation of m/z 906.81 (RT 7.2), 912.15 (RT 7.0), 864.79 (RT 7.5) and 960.51 (RT 5.9) found in Palythoa canariensis. A, B, C and D indicate the main peaks of each m/z. B) Chromatographic separation of m/z 906.81, 912.15 and 864.79 in the PLTX commercial standard (Palythoa tuberculosa, Wako). C) Chromatographic separation of m/z 906.81 and 912.15 in the Palythoa tuberculosa extract.


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Figure 3. Ionization and fragmentation spectra of the molecule eluting at RT 7.2 min in Palythoa canariensis (molecule A, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bi-; iv and v) monocharged and vi) Amoiety ions. B) Segments of MS2 spectrum obtained from the fragmentation of the m/z 906.81 tricharged ion: i) m/z 300-650; ii) m/z 700-905 and iii) m/z 1050-1225. 160x137mm (300 x 300 DPI)



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Figure 4. Ionization and fragmentation spectra of the molecule eluting at RT 7.0 min in Palythoa canariensis (molecule B, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bicharged; and iv) monocharged ions. B) Segments of MS2 spectrum obtained after fragmentation of the m/z 912.15 tricharged ion: i) m/z 300-660; ii) m/z 700-915 and iii) m/z 1125 to 1195. 180x111mm (300 x 300 DPI)


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Figure 5. Ionization and fragmentation spectra of the molecule eluting at RT 7.5 min in Palythoa canariensis (molecule C, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bicharged; and iv) monocharged ions. B) Segments of MS2 spectrum obtained after fragmentation of the m/z 864.79 tricharged ion: i) m/z 300-670; ii) m/z 640-880 and iii) m/z 1000-1500. 180x103mm (300 x 300 DPI)



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Figure 6. Ionization and fragmentation spectra of the molecule eluting at RT 5.9 min in Palythoa canariensis (molecule D, Figure 2A). A) MS1 spectrum showing i) tri-; ii and iii) bicharged ions. B) Segments of MS2 spectrum obtained from the fragmentation of the m/z 960.51 tricharged ion: i) m/z 300-660; ii) m/z 8801000 and iii) m/z 1180-1280. 169x72mm (300 x 300 DPI)


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First Identification of Palytoxin-Like Molecules in the Atlantic Coral

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