Identification of the Photosynthetic Pigments of the Tropical Benthic 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. , 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 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 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 present. The major 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 , , and B­ GPM medium (Loeblich, 1975) adjusted Pigment fractions were then dissolved . Gambierdiscus toxicus also con­ to 31°/00 salinity in 2.8 liter Fernbach in ethanol or acetone. An absorption tained a water soluble -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 , 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 of this (1985) found no evidence for the pres­ (Table 2). lineage. Those species (belonging to the ence of an internal symbiont as an The two major yellow peridinioid lineage) that harbor a photo­ explanation for the occurrence of the produced by this organism had nearly synthetic endosymbiont (Jeffrey et aI., second form of chlorophyll c. There re­ identical spectral properties and Rf 1975) are atypical as some of the pig­ mains the possibility that the fraction values (Table 1, Fig. 1). The first of the ments may belong to the symbiont de­ they identify as "chlorophyll-cl" is a two to develop during chromatographic rived from a different algal division: chlorophyll degradation product that separation was yellow-orange and par­ e.g., in balticum could result from photooxidation. Such titioned between hexane and 95 percent (Tomas and Cox, 1973). Jeffrey et aI. degradation products may occur if pig­ methanol in the ratio of 5:95 (hexane: (1975) noted that all of the peridinin con­ ments are not analyzed under reduced methanol) (Table 2). This pigment has taining photosynthetic dinoflagellate light conditions and in a nonoxidizing been identified as diadinoxanthin. The species studied contained an unknown (nitrogen) atmosphere. The discrepan­ second to elute was bright "pink" pigment, which remained at the cies between the pigment pattern for the yellow and had a partition coefficient origin during thin layer chromatography. Florida and Pacific isolates of G. tox­ ratio of 6:94 (hexane:methanol) and has Thin-layer chromatography of G. tox­ icus suggest that it may be necessary to been identified as dinoxanthin. Based on icus pigments revealed this same frac­ analyze more isolates before a clear densitometric scan data, both xantho­ tion (1), although in G. toxicus this pig­ understanding ofthe apparent variability phylls are found in approximately equal ment was brown. Spectral data and im­ can be reconciled. amounts in the cell, together compos­ mobility in a nonpolar solvent suggest Durand and Berkaloff (1985) reported ing 9.8 percent ofthe total pigment con­ this to be the peridinin-chlorophyll-a only two carotenoids, the xanthophylls tent and 'D.2 percent of the total carot­ protein complex. These protein-pigment diadinoxanthin and peridinin, from the enoids of G. toxicus by weight (Table 2). complexes act in a light harvesting capa­ Pacific G. toxicus; their study dealt See Table 3 for the carotenoid and city (Prezelin and Haxo, 1976) and ap­ mainly with the ultrastructure and chlorophyll pigment ratios on a weight pear to be an integral part of the dino­ cWorophyll pigmentation. No and molar basis. flagellate photosynthetic apparatus. were reported for the Pacific isolate. These photosynthetic complexes have The apparent differences in the carote­ been observed in other dinoflagellates noid pigmentation between the Florida such as Glenodinium sp. (Prezelin, and Pacific isolates may disappear when

Table 3.-Pigment ratios for Gambierd/scus fox/cus. 1976), polyedra (Prezelin a more detailed analysis of the Pacific and Haxo, 1976), carterae form is published. WI. Mol. Pigments ratio ratio (Haxo et aI., 1976; Siegelman et aI., The properties offractions 2 and 3 are 1976), furca (Meeson et aI., similar to pheophytin-a, a magnesium­ Chlorophyll-a:Chlorophyll-e, 2.91 1.98 Total chlorophyll:Total carotenoid 1.77 2.82 1982), and Heterocapsa spp. (Watson deficient chlorophyll molecule, which Peridinin:Chlorophyll-a 0.48 0.68 Total carotenoid:Chlorophyll-a 0.76 and Loeblich, 1983). has been reported in Peridinium cinc­ Durand and Berkaloff (1985) reported tum (Strain et aI., 1944) and in Pacific the presence of both cWorophyll-Cl and Gyre phytoplankton samples (Jeffrey, chlorophyll-C2 in G. toxicus. Our re­ 1975). It is not known whether pheo­ sults disagree as we found only chloro­ phytin-a occurs naturally or if it is a Occasionally, after centrifugation, phyll-C2. Presence of chlorophyll-cl laboratory artifact. freeze-thawing, or filtration of G. tox­ and C2 in dinoflagellates whose major Jeffrey et aI. (1975), in a survey of icus cells, an orange water-soluble pig­ carotenoid is peridinin has been seen in dinoflagellate pigments, showed a range ment appeared in the supernatant. From only one species, Prorocentrum cassu­ for peridinin, the major dinophycean spectral data, this orange pigment has bicum (Jeffrey, 1976); all other photo­ carotenoid, of 54-68 percent of the total been identified as a peridinin-chloro­ synthetic dinoflagellates have only carotenoid fraction. The value of64 per­ phyll-a protein complex. chlorophyll a and chlorophyll-C2. The cent which we recorded for G. toxicus absence of a second form of cWorophyll is within this range. Similar results ex­ Discussion c in our isolate, and the lack of other ist for the ratio of peridinin to chloro­ Comparison of our data concerning reports of this pigment in any dinoflagel­ phyll-a and for the ratio of total carote­ the chloroplast pigment composition of late that is morphologically related to G. noids to chlorophyll-a where ranges of

46 Marine Fisheries Review 0.32-0.50 and 0.60-0.74 are found, Lond., 356 p. Prezelin, B. B. 1976. The role of peridinin-chloro­ Haxo, F. T., 1. H. Kycia, G. F. Somers, A. Ben­ phyll a-proteins in the photosynthetic light adap­ respectively. Gambierdiscus toxicus ex­ nett, and H. W. Seigelman. 1976. Peridinin­ tation ofthe marine dinoflagellate, Glenodinium hibits values of 0.48 for peridinin: chlorophyII a proteins of the dinoflagellate sp. Planta (Berl.). 130:225-233. chlorophyll-a and 0.76 for total carote­ Amphidinium carterae (Plymouth 450). Plant ____ , and F. T. Haxo. 1976. Purification Physiol. 57:297-303. and characterization of peridinin-chlorophyll a­ noids:chlorophYll-a (Table 3). Jeffrey, S. W. 1975. Green algal pigments in the proteins from the marine dinoflagellates Gleno­ Although the relative percentages of central north Pacific Ocean. In CSIRO Mar. dinium sp. and Gonyaulax polyedra. Planta Biochem. Unit Annu. Rep. 1974-1975, p. 23-25. (Berl.). 128:133-141. pigments may vary from species to spe­ ____ . 1976. The occurrence of chlorophyll Riley, 1. P., and T. R. S. Wilson. 1967. The pig­ cies, the basic components of the dino­ c! and C2 in algae. 1. Phycol. 12:349-354. ments of some marine phytoplankton species. flagellate photosynthetic apparatus are ____ , M. Sielicki, and F. T. Haxo. 1975. 1. Mar. BioI. Assoc. U.K. 47:351-362. Chloroplast pigment patterns in dinoflagellates. Siegelman, H. w., 1. H. Kycia, and F. T. Haxo. present in all species for which data is 1. Phycol. 11:374-384. 1976. Peridinin-chlorophyll a-proteins of dino­ available; such is the case with G. tox­ ____ ,1. Ulrich, and M. B. Allen. 1966. flagellate algae. In Chlorophyll-proteins, reac­ icus. See Jeffrey et al. (1975) for a re­ Some photochemical properties of chloroplast tion centers, and photosynthetic membranes. preparations from the chrysomonad Hymeno­ Brookhaven Symp. BioI. 28:162-169. view of dinoflagellate pigmentation. monas sp. Biochim. Biophys. Acta. 112:35­ Strain, H. H., W. M. Manning, and G. Hardin. 44. 1944. Xanthophylls and carotenes of diatoms, Loeblich, A. R., III. 1975. A seawater medium brown algae, dinoflagellates, and sea-anem­ Acknowledgments for dinoflagellates and the nutrition of Cacho­ ones. BioI. Bull. 86:169-191. nina niei. 1. Phycol. 11:80-86. Tomas, R. N., and E. R. Cox. 1973. The symbio­ The authors thank Alfred R. Loeblich ____ , and S. R. Indelicato. 1986. Thecal sis of Peridinium balticum () 1. III and Robert leBoeuf for their help­ analysis of the tropical benthic dinoflagellate Ultrastructure and pigment analysis. 1. Phycol. ful comments and suggestions. This re­ Gambierdiscus toxicus. Mar. Fish. Rev. 48(4): 9 (suppl.):16. 38-43. Watson, D. A., and A. R. Loeblich III. 1983. An search was funded in part by a Univer­ Meeson, B. w., S. S. Chang, and B. M. Sweeney. application of electrophoresis to the systema­ sity of Houston Coastal Center Grant. 1982. Characterization of peridinin-chlorophyll tics of the marine dinoflagellate genus Hetero­ a-proteins from the marine dinoflagellate Cera­ capsa. Biochem. System. Ecol. 11:67-71. tium furca. Bot. Mar. 25:347-350. Yasumoto, T., I. Nakajima, R. Bagnis, and R. Literature Cited Norgard, S., W. A. Svec, S. Liaaen-Jensen, A. Adachi. 1977. Finding a dinoflagellate as a likely Jensen, and R. R. L. Guillard. 1974. Chloro­ culprit of ciguatera. Bull. Jpn. Soc. Sci. Fish. Durand, M., and C. Berkaloff. 1985. Pigment plast pigments and algal systematics. Biochem. 43:1021-1026. composition and chloroplast organization of System. Ecol. 2:3-6. -c:-----,-- , , y. Oshima, and R. Gambierdiscus toxicus Adachi and Fukuyo Petracek, F. 1., and L. Zechmeister. 1956. Deter­ Bagnis. 1979. A new toxic dinoflagellate found (Dinophyceae). Phycologia 24:217-223. mination of partition coefficients ofcarotenoids in association with ciguatera. In D. L Taylor Goodwin, T. W. 1952. The comparative biochem­ as a tool in pigment analysis. Anal. Chern. and H. H. Seliger (editors), Toxic dinoflagel­ istry of the carotenoids. Chapman and Hall, 28:1484-1485. late blooms, p. 65-70. Elsevier Sci. Publ., NY.

48(4), 1986 47