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Identification of the Photosynthetic Pigments of the Tropical Benthic Dinoflagellate Gambierdiscus Toxicus

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Identification of the Photosynthetic Pigments of the Tropical Benthic Dinoflagellate Gambierdiscus Toxicus Identification of the Photosynthetic Pigments of the Tropical Benthic Dinoflagellate Gambierdiscus toxicus STEPHEN R. INDELICATO and DAVID A. WATSON Introduction in comparative studies with other dino­ explains culture procedures and condi­ flagellate species. Pigment composition tions. Gambierdiscus toxicus, a marine ben­ of microalgae has been used as a tax­ Cultures were harvested by continu­ thic dinoflagellate, is currently of inter­ onomic criterion for a number of years ous-flow centrifugation at the end ofthe est to toxicologists since it has been (Strain et al., 1944; Goodwin, 1952; exponential phase of growth. The result­ found to produce toxins that have been Riley and Wilson, 1967; Norgard et al., ing cell pellet was sonicated in acetone implicated in ciguatera poisoning (Yasu­ 1974). Studies on chloroplast pigment and periodically shaken to facilitate the moto et al., 1977, 1979). While numer­ patterns of photosynthetic dinoflagel­ extraction of the chloroplast pigments. ous reports have focused on the struc­ lates have assisted biologists in group­ The pigment-containing acetone extract ture and mechanism of action of the ing these organisms on biochemical data was repeatedly drawn off the cell debris, toxins associated with G. toxicus, rela­ (Jeffrey et al., 1975) in addition to the and fresh acetone was added until the tively few have addressed fundamental classical groupings based on morphol­ acetone fraction was nearly colorless. questions regarding the nontoxin ogy. The acetone extract was then briefly biochemistry and physiology of this There has been one published study centrifuged to remove particulate cell organism. on the cWoroplast ultrastructure coupled debris from the preparation and evapor­ In this paper we add additional data with data for some of the pigments of ated to dryness under a stream of nitro­ that can be used by others in character­ G. toxicus (Durand and Berkaloff, gen at less than 40°C. izing their G. toxicus strains as well as 1985). In their study, however, the carot­ Dried pigments were dissolved in 180 enoids were not completely character­ Jll of carbon disulfide and spotted re­ ized, either qualitatively or quantitative­ peatedly onto activated silica gel thin­ ly. Additionally, they report the unusual layer chromatographic (TLC) plates, occurrence of chlorophyll Cl. In this using 20 Jll micropipettes. Development ABSTRACT-Photosynthetic pigments of paper we identify the major photosyn­ took place in a mixture of hexane/ace­ the Floridn isolate ofGambierdiscus toxicus thetic pigments ofthe Florida isolate of tone (6:4) in a sealed chamber. were investigated to aid in characterizing this G. toxicus and compare them with the Developed plates were scanned at 470 strain and to assist in comparisons with data for the Pacific strain ofthis species. nm using a Helena Quick-scan R&D Pacific Ocean isolates. The pigments were scanning densitometer. The readout was separated using thin-layer chromatography Materials and Methods (FLC). Tentative pigment identifications were used to provide an accurate means of madefrom visible absorption maxima (in two Gambierdiscus toxicus was isolated locating the center ofeach pigment band solvents) and partition coefficients (hexane: from an intertidal environment on the to aid in the calculation of Rf values. 95 percent methanol). The TLC revealed the southern coast of Florida by A. R. The densitometer integrated the areas presence of10 pigment bands. The chloro­ Loeblich III in 1983 and designated under the peaks from which relative phylls a and C2 were the major chlorophylls present. The major carotenoid was peridi­ strain F8. Strain F8 was later grown for percentages for each carotenoid were nin, followed in abundnnce on a weight basis pigment analysis in 1.5 liter batches of calculated. by diadinoxanthin, dinoxanthin, and B­ GPM medium (Loeblich, 1975) adjusted Pigment fractions were then dissolved carotene. Gambierdiscus toxicus also con­ to 31°/00 salinity in 2.8 liter Fernbach in ethanol or acetone. An absorption tained a water soluble peridinin-chlorophyll l a-protein complex. A trichromatic method flasks . The previous paper in this con­ spectrum for each pigment fraction was was used to quantify the amount of total ference (Loeblich and Indelicato, 1986) produced over the visible light range carotenoids, chlorophyll a, and chlorophyll c. The Floridn isolate ofthis species differs from the published data for the Pacific isolate ofthis species in having only the C2 form of 'Reference to trade names or commercial firms The authors are with the Marine Science Program, chlorophyll c and qualitatively more carote­ does not imply endorsement by the National University of Houston, 4700 Avenue U, Galves­ noids. Marine Fisheries Service, NOAA. ton, TX 77551. 44 Marine Fisheries Review Table 1.-Gamblardiscus toxicus chloroplast pigment R, valuea and absorb­ tlon maxima. 1.0 ------------- R, value2 (acetone: Absorption maxima' CJ- J3-car Pigment' Color hexane) (ethanol) (acetone) Origin Brown 0.00 454, 590, 665 No data - Chlorophyll-c Grassilreen 0.09 445, 587, 636 450, 583, 633 Q-(Chl-a) Unknown Brown-green 0.35 No data c::::::J -­ 0- Chl-a Peridinin Red-orange 0.44 473 g-Din 470 0.5- -- Diad Diadinoxanthin Yellow-orange 0.51 '(409), 431, 457 CJ) 0 ____ (4t3), 438, 460 w Dinoxanthin Yellow 0.52 (405), 429, 457 ::::> (404), 429, 456 ...J Chlorophyll-a Green 0.56-0.57 413. 504, 535, 615, 666 « 412. 505. 535. 615. 666 > (Chlorophyll-a) Gray-green 0.62 410. 506. 535. 613. 669 LL No data a:: Q-Chl-C (Chlorophyll-a) Gray-green 0.64-0.66 413,510,540.612.670 o Origin 412,507.536.610.668 0'-- B-carotene Yellow 0.91-0.98 429. 451, 478 (404), 429. 453, 475 'Pigments are listed in order of increasing mobility. 2These values were determined using a developing solvent consisting of 40 parts acetone and 60 parts hexane. Figure I.-Representation of a thin-layer chroma­ 'For each pigment. the absorbtion maxima as measured in ethanol are on the tographic plate showing separation of Gambier­ first line and the absorbtion maxima as measured in acetone are on the second line. discus toxicus photosynthetic pigments. 4Absorption maxima given in parentheses are values for shoulders in the spec­ trum which could not be defined as a clear peak. (350-750 nm) using a Beckman model The major pigment within the cells of Table 2.-Percent total pigments, parcent total carote­ nolds, and carotenoid partition coefficients tor Gam­ 35 spectrophotometer. Partition coeffi­ G. toxicus as measured by percent com­ biardiscus toxiCUB. cient values were determined, using the position is chlorophyll-a (fraction 4) Percent Percent Partition method of Petracek and Zechmeister (Table 2). Spectral properties of two total total coefficient pig- carot- (hexane: (1956), to aid in the identification of other fractions (2, 3), which are gray­ Pigment ments enoids acetone) pigments. The trichromatic method of green and have slightly higher R values f Chlorophyll-a 47.6 Jeffrey et al. (1966) was employed to ob­ than chlorophyll-a, suggest that these Chlorophyll-c2 16.3 tain values for the relative weight per­ are degradation products of chlorophyll­ Peridinin 23.0 63.6 3:97 Diadinoxanthin 4.9 13.6 5:95 centages ofcarotenoids and chlorophylls a. Together, chlorophyll-a and its two Dinoxanthin 4.9 13.6 6:94 present in a whole-cell extract (95 per­ degradation products make up 47.6 per­ B-Carotene 3.3 9.1 cent acetone) prepared from the cells of cent by weight of the total pigments in G. toxicus. this species (Table 2). Chlorophyll-C2 (fraction 9) is also Results found in large amounts in G. toxicus Thin layer chromatography revealed cells, constituting 16.33 percent of the the presence of 10 pigment fractions ex­ total pigment weight (Table 2). This pig­ nin constituted 23.0 percent of the total tracted from the cells of G. toxicus (Fig. ment's color is grass-green and was far cellular pigments and 63.6 percent of the 1). Of these 10 fractions (numbered in less mobile than chlorophyll-a when total carotenoids by weight (Table 2). order of elution), four had obvious chromatographically developed in an Peridinin is easily recognized by its chlorophyll affinities (fractions 9, 4, 3, acetone:hexane solvent (Fig. 1). There bright red-orange color and its charac­ 2), four were carotenoids (fractions 7, was no evidence of chlorophyll-cl in teristic broad absorption maximum at 6, 5, 1), and two others (fractions 8, 10) this strain of G. toxicus. around 473 nm. It is the last carotenoid were unknown. The chromatographic Ofthe four carotenoid pigments found to be eluted during chromatographic and spectral properties ofthese fractions in G. toxicus, peridinin (fraction 7) was separation in an acetone:hexane solvent are presented in Table 1. present in the greatest amount. Peridi- (Fig. 1). 48(4), 1986 45 A yellow pigment fraction, which was G. toxicus with that of other dinoflagel­ toxicus, suggests strongly that the report the most mobile of all pigments con­ late species reveals that G. toxicus by Durand and Berkaloff (1985) should tained in G. toxicus, and which traveled possesses a pigment content which is be reconfirmed. with the solvent front (Rfvalue = 0.91­ very similar to that of other dinoflagel­ Additionally, P. cassubicum belongs 0.98), was identified as B-carotene. Of lates belonging to the gonyaulacoid lin­ to a dinoflagellate lineage that shows af­ the major pigments of G. toxicus, B­ eage. Chlorophyll-a, chlorophyll-C2, finities to the dinophysioids rather than carotene was found to constitute only 9.1 peridinin, dinoxanthin, diadinoxanthin, to the gonyaulacoids to which G. tox­ percent of the total carotenoids and 3.3 and B-carotene have been found in all icus belongs. Durand and Berkaloff percent of the total pigment content photosynthetic dinoflagellates of this (1985) found no evidence for the pres­ (Table 2).
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