A SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS,KARENIA MIKIMOTOI,AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE1

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4‐methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (ED) and its 27‐nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.     

Molecular detection of the brevetoxin‐producing dinoflagellate Karenia brevis and closely related species using rRNA‐targeted probes and a semiautomated sandwich hybridization assay1

Brevetoxins produced by the marine dinoflagellate Karenia brevis (C. C. Davis) G. Hansen et Moestrup cause neurotoxic shellfish poisoning (NSP) in human consumers and also endanger a variety of coastal wildlife. In the eastern Gulf of Mexico the presence and abundance of this species have traditionally been monitored using light microscopy (LM) observations of whole water samples. Various molecular probe methods now enable detection of multiple species from a single sample, allowing rapid sample analysis. We describe the development of sandwich hybridization assays (SHAs) for Karenia brevis, K. selliformis Haywood, Steid. et L. MacK., K. mikimotoi (Miyake et Kominami ex M. Oda) G. Hansen et Moestrup, K. papilionacea Haywood et Steid., the Karlotoxin‐producer Karlodinium veneficum (D. Ballant.) J. Larsen (=K. micrum), and Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov. The assays require no nucleic acid purification and use LSU rRNA‐targeted probes and a semiautomated, 96‐well plate format. Probes tested in matrix format were specific relative to rRNAs of all nontarget species used. The response of the SHA for a constant number of K. brevis cells per unit volume of homogenate depended on the growth status of a culture, decreasing for senescent cells relative to actively growing cells. The results of preliminary field tests of the K. brevis SHA indicated that cells collected from natural populations tended to return a lower signal than those harvested from laboratory cultures, but these results are nonetheless very encouraging. These preliminary field studies show that robust standards are required for cell identification and enumeration, with which new methods can be compared.     

TAKAYAMA GEN. NOV. (GYMNODINIALES,DINOPHYCEAE), A NEW GENUS OF UNARMORED DINOFLAGELLATES WITH SIGMOID APICAL GROOVES,INCLUDING THE DESCRIPTION OF TWO NEW SPECIES1

A new potentially ichthyotoxic dinoflagellate genus, Takayama de Salas, Bolch, Botes et Hallegraeff gen. nov., is described with two new species isolated from Tasmanian (Australia) and South African coastal waters: T. tasmanica de Salas, Bolch et Hallegraeff, sp. nov. and T. helix, de Salas, Bolch, Botes et Hallegraeff, sp. nov. The genus and two species are characterized by LM and EM of field samples and laboratory cultures as well as large subunit rDNA sequences and HPLC pigment analyses of several cultured strains. The new Takayama species have sigmoid apical grooves and contain fucoxanthin and its derivatives as the main accessory pigments. Takayama tasmanica is similar to the previously described species Gymnodinium pulchellum Larsen, Gyrodinium acrotrochum Larsen, and G. cladochroma Larsen in its external morphology but differs from these in having two ventral pores, a large horseshoe‐shaped nucleus, and a central pyrenoid with radiating chloroplasts that pass through the nucleus. It contains gyroxanthin‐diester and a gyroxanthin‐like accessory pigment, both of which are missing in T. helix. Takayama helix has an apical groove that is nearly straight while still being clearly inflected. A ventral pore or slit is present. It has numerous peripheral, strap shaped, and spiraling chloroplasts with individual pyrenoids and a solid ellipsoidal nucleus. The genus Takayama has close affinities to the genera Karenia and Karlodinium.     

LIPID,FATTY ACID,AND STEROL COMPOSITION OF EIGHT SPECIES OF KARENIACEAE (DINOPHYTA): CHEMOTAXONOMY AND PUTATIVE LIPID PHYCOTOXINS1

The lipid class, fatty acid, and sterol composition of eight species of ichthyotoxic marine gymnodinioid dinoflagellate (Karenia, Karlodinium, and Takayama) species was examined. The major lipid class in all species was phospholipid (78%–95%), with low levels of triacylglycerol (TAG; 0%–16%) and free fatty acid (FFA; 1%–11%). The common dinoflagellate polyunsaturated fatty acids (PUFA), octadecapentaenoic acid (OPA 18:5ω3), and docosahexaenoic acid (DHA 22:6ω3), were present in all species in varying amounts (14%–35% and 8%–23%, respectively). The very‐long‐chain PUFA (VLC‐PUFA) 28:7ω6 and 28:8ω3 were present at low levels (<1%), and the ratio of these fatty acids may be a useful chemotaxonomic marker at the species level. The typical dinoflagellate sterol dinosterol was absent from all species tested. A predominance of the 4‐methyl and 4‐desmethyl Δ⁸⁽¹⁴⁾ sterols in all dinoflagellate species included 23‐methyl‐27‐norergosta‐8(14),22‐dien‐3β‐ol (Karenia papilionacea A. J. Haywood et Steid, 59%–66%); 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, brevesterol, (Takayama tasmanica de Salas, Bolch et Hallegraeff 84%, Takayama helix de Salas, Bolch, Botes et Hallegraeff 71%, Karenia brevis (C. C. Davis) G. Hansen et Moestrup 45%, Karlodinium KDSB01 40%, Karenia mikimotoi (Miyake et Kominami ex Oda) G. Hansen et Moestrup 38%); and (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, gymnodinosterol, (K. mikimotoi 48%, Karenia umbella de Salas, Bolch et Hallegraeff 59%, Karlodinium veneficum (D. L. Ballant.) J. Larsen 71%–83%). In Takayama species, five steroid ketones were identified, including for the first time the 3‐keto form of brevesterol and gymnodinosterol. These results indicate a biochemical link between sterol and steroid ketone biosynthesis, suggesting that selected dinoflagellates can make a significant contribution to ketones in marine sediments. The presence of steroid ketones, specific sterols, and fatty acids, and the ratio of VLC‐PUFA may prove to be a useful chemotaxonomic tool for distinguishing between morphologically similar species. The relative levels of the PUFA, OPA, and DHA, coupled with the potential inhibitory action of Δ⁸⁽¹⁴⁾ sterols, may provide an insight into the ichthyotoxicity of these bloom‐forming dinoflagellates.     

Effect of salinity on the distribution, growth, and toxicity of Karenia spp.

The first recorded bloom of Karenia spp., resulting in brevetoxin in oysters, in the low salinity waters of the Northern Gulf of Mexico (NGOMEX) occurred in November 1996. It raised questions about the salinity tolerance of Karenia spp., previously considered unlikely to occur at salinities <24 psu, and the likelihood that the bloom would reoccur in the NGOMEX. Salinity was investigated as a factor controlling Karenia spp. abundance in the field, using data from the NGOMEX 1996 bloom and Florida coastal waters from 1954 to 2004, and growth and toxin production in cultures of Karenia brevis (Davis) G. Hansen and Moestrup. During the NGOMEX bloom, Karenia spp. occurred much more frequently at low salinities than in Florida coastal waters over the last 50 years. The data suggest that the NGOMEX bloom started on the NW Florida Shelf, an area with a higher frequency of Karenia spp. at low salinities than the rest of Florida, and was transported by an unusual westward surface current caused by Tropical Storm Josephine. The minimum salinity at which growth occurred in culture ranged between 17.5 and 20 psu, but the optimal salinity ranged between low values of 20 or 25 and high values of 37.5–45 psu, depending on the clone. The effect of salinity on toxin production in one clone of K. brevis was complex, but at all salinities brevetoxin levels were highest during the stationary growth phase, suggesting that aging, high density blooms may pose the greatest public health threat. The results demonstrate that Karenia spp. can be a public health threat in low salinity areas, but the risk in the NGOMEX is relatively low. No bloom has occurred since the 1996 event, which was probably associated with a special set of conditions: a bloom along the Florida Panhandle and a tropical storm with a track that set up a westward current.     

QUANTIFICATION OF THE RELATIVE ABUNDANCE OF THE TOXIC DINOFLAGELLATE,KARENIA BREVIS (DINOPHYTA), USING UNIQUE PHOTOPIGMENTS1

Diagnostic photopigment analysis is a useful tool for determining the presence and relative abundance of algal groups in natural phytoplankton assemblages. This approach is especially useful when a genus has a unique photopigment composition. The toxic dinoflagellate Karenia brevis (Davis) G. Hansen & Moestrup comb. nov. shares the diagnostic pigment gyroxanthin‐diester with only a few other dinoflagellates and lacks peridinin, one of the major diagnostic pigments of most dinoflagellate species. In this study, measurements of gyroxanthin‐diester and other diagnostic pigments of K. brevis were incorporated into the initial pigment ratio matrix of the chemical taxonomy program (CHEMTAX) to resolve the relative contribution of K. brevis biomass in mixed estuarine phytoplankton assemblages from Florida and Galveston Bay, Texas. The phytoplankton community composition of the bloom in Galveston Bay was calculated based on cell enumerations and biovolumetric measurements in addition to chl a‐specific photopigment estimates of biomass (HPLC and CHEMTAX). The CHEMTAX and biovolume estimates of the phytoplankton community structure were not significantly different and suggest that the HPLC–CHEMTAX approach provides reasonable estimates of K. brevis biomass in natural assemblages. The gyroxanthin‐diester content per cell of K. brevis from Galveston Bay was significantly higher than in K. brevis collected from the west coast of Florida. This pigment‐based approach provides a useful tool for resolving spatiotemporal distributions of phytoplankton in the presence of K. brevis blooms, when an appropriate initial ratio matrix is applied.     

A modified assay to determine hemolytic toxin variability among Karenia clones isolated from the Gulf of Mexico

Karenia brevis (formerly Gymnodinium breve) is a toxic marine dinoflagellate generally restricted to the Gulf of Mexico and is the main causative organism in fish kills, shellfish intoxications and respiratory distress in humans following bloom events. K. mikimotoi is a morphologically similar co-occurring species which is toxic in other parts of the world oceans, but has not been recognized as a major contributor in toxicity of blooms within the Gulf of Mexico. Recently there has been increasing evidence of the simultaneous production of a variety of bioactive compounds in addition to potent neurotoxins (brevetoxin) in Karenia brevis isolates. These compounds are potentially ichthyotoxic and have been shown to cause hemolysis in several bioassays [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519; Kirkpatrick, B., Fleming, L.E., Squicciarini, D., Backer, L.C., Clark, R., Abraham, W., Benson, J., Cheng, Y.S., Johnson, D., Pierce, R., Zaias, J., Bossart, G.D., Baden, D.G., 2004. Literature review of Florida red tide: implications for human health effects. Harmful Algae 3, 99–115]. Presence of hemolytic compounds may therefore add to the overall toxicity levels of bloom events. Current monitoring methods include assays which are highly sensitive in brevetoxin detection and yet may not target other harmful compounds.By adapting protocols developed by Eshbach et al. [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519], Red drum (Sciaenops ocellatus) erythrocytes were used to create a modified bioassay to detect hemolytic activity of crude algal extracts. Red drum was selected because it is endemic to coastal areas throughout the Gulf of Mexico and is sensitive to Karenia blooms, and thus makes this species a valid ecological target. Preliminary data has shown this method is sensitive for use in assessing hemolysis induced by laboratory cultures down to levels of 1 × 10³ cells mL⁻¹. Results showed an unexpectedly high level of hemolytic activity among K. mikimotoi clones, with one Texas strain inducing significantly higher hemolysis compared to Florida K. brevis isolates. Using this approach, future research efforts will examine the difference in production of hemolytic compounds among various Karenia clones.     

Galactolipid composition of the star-shaped dinoflagellate Asterodinium gracile (Kareniaceae): presence of hexadecatetraenoic acid (16:4(n-3))-containing monogalactosyldiacylglycerol as the predominant galactolipid and chemotaxonomic closeness to Karenia mikimotoi as the only other known Kareniacean producer of this fatty acid

Asterodinium gracile is a morphologically distinct, star-shaped member of the Kareniaceae with, like canonical Kareniaceae, a tertiary plastid of haptophyte origin. However, A. gracile's complement of carotenoid photosynthetic pigments has been shown to be chemotaxonomically atypical in that it possesses much less fucoxanthin when compared to that of other, canonical Kareniaceae in the genera Karenia, Karlodinium, and Takayama, also with a tertiary plastid of haptophyte origin. To date, Karenia mikimotoi, Karenia papilionacea, and Karenia selliformis are the only canonical Kareniaceae that have been shown to have a chemotaxonomically atypical carotenoid pigment composition in that they possess a gyroxanthin diester-like carotenoid not observed in other species of Karenia, Karlodinium, or Takayama (recognizing that Karenia, in general, produces fucoxanthin derivatives not observed in Karlodinium or Takayama). As a photosynthetic organism, K. mikimotoi has been shown to resemble Karenia brevis such that both species possess the chloroplast-associated galactolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) enriched with octadecapentaenoic acid (18:5(n-3)) in the sn-1 position, and hexadecenoic acid (16:0) and tetradecanoic acid (14:0) at the sn-2 position. However, K. mikimotoi is chemotaxonomically atypical beyond its carotenoid composition in that it possesses MGDG and DGDG with hexadecatetraenoic acid (16:4(n-3)), which has not been observed in any other members of the Kareniaceae, in the sn-2 position as major galactolipids. The goal of this study was to characterize the galactolipids of A. gracile with the hypothesis that they would also be atypical when compared to other canonical Kareniaceae because of A. gracile's atypical carotenoid pigment composition. To this end, we report that like K. brevis and K. mikimotoi, A. gracile produces MGDG and DGDG enriched in 18:5(n-3) at the sn-1 position and C₁₄ fatty acids, such as 14:0, at the sn-2 position, and like K. mikimotoi, it produces 18:5(n-3)/16:4(n-3) MGDG, yet here as its most abundant galactolipid.     

A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia brevis in the Rookery Bay National Estuarine Research Reserve

A DNA hybridization assay was developed in microtiter plate format to detect the presence of toxic dinoflagellates in coastal waters. Simultaneous detection of multiple species was demonstrated using Karenia brevis, Karenia mikimotoi, and Amphidinium carterae. Molecular probes were designed to detect both K. brevis and K. mikimotoi and to distinguish between these two closely related species. The assay was used to detect K. brevis in coastal waters collected from the Rookery Bay National Estuarine Research Reserve. Assay results were verified by species-specific PCR and sequence analysis. The presence/absence of K. brevis was consistent with microscopic observation. Assay sensitivity was sufficient to detect K. brevis in amounts defined by a regional monitoring program as “present” (≤1000 cells/L). The assay yielded quick colorimetric results, used a single hybridization temperature, and conserved the amount of genomic DNA utilized by employing one set of PCR primers. The microplate assay provides a useful tool to quickly screen large sample sets for multiple target organisms.     

MORPHOLOGY AND MOLECULAR ANALYSES OF THREE TOXIC SPECIES OF GAMBIERDISCUS (DINOPHYCEAE): G. PACIFICUS, SP. NOV., G. AUSTRALES, SP. NOV., AND G. POLYNESIENSIS, SP. NOV.

Three new dinoflagellate species, Gambierdiscus polynesiensis, sp. nov., Gambierdiscus australes, sp. nov., and Gambierdiscus pacificus, sp. nov., are described from scanning electron micrographs. The morphology of the three new Gambierdiscus species is compared with the type species Gambierdiscus toxicus Adachi et Fukuyo 1979, and two other species: Gambierdiscus belizeanus Faust 1995 and Gambierdiscus yasumotoi Holmes 1998. The plate formula is: Po, 3′, 7", 6C, 8S, 5‴, 1p, 2". Culture extracts of these three new species displayed both ciguatoxin- and maitotoxin-like toxicities. The following morphological characteristics differentiated each species. 1) Cells of G. polynesiensis are 68–85 μm long and 64–75 μm wide, and the cell’s surface is smooth. They are identified by a large triangular apical pore plate (Po), a narrow fish-hook opening surrounded by 38 round pores, and a large, broad posterior intercalary plate (1p) wedged between narrow postcingular plates 2‴ and 4‴. Plate 1p occupies 60% of the width of the hypotheca. 2) Cells of G. australes also have a smooth surface and are 76–93 μm long and 65–85 μm wide in dorsoventral depth. They are identified by the broad ellipsoid apical pore plate (Po) surrounded by 31 round pores and a long and narrow 1p plate wedged between postcingular plates 2‴ and 4‴. Plate 1p occupies 30% of the width of the hypotheca. 3) Cells of G. pacificus are 67–77 μm long and 60–76 μm wide in dorsoventral depth, and its surface is smooth. They are identified by the four-sided apical pore plate (Po) surrounded by 30 round pores. A short narrow 1p plate is wedged between the wide postcingular plates 2‴ and 4‴. Plate 1p occupies 20% of the width of the hypotheca. These three newly described species were also characterized by isozyme electrophoresis and DNA sequencing of the D8–D10 region of their large subunit (LSU) rRNA genes. The consistency between species designations based on SEM microscopy and classification inferred from biochemical and genetic heterogeneities was examined among seven isolates of Gambierdiscus. Their classification into four morphospecies was not consistent with groupings inferred from isozyme patterns. Three molecular types could be distinguished based on the comparison of their LSU rDNA sequences. Although G. toxicus TUR was found to be more closely related to G. pacificus, sp. nov. than to other G. toxicus strains, the molecular classification was able to discriminate G. polynesiensis, sp. nov. and G. australes, sp. nov. from G. toxicus. These results suggest the usefulness of the D8–D10 portion of the Gambierdiscus LSU rDNA as a valuable taxonomic marker.     

The potential threat of algal blooms to the abalone (Haliotis midae) mariculture industry situated around the South African coast

Toxic algal blooms are common world-wide and pose a serious problem to the aquaculture and fishing industries. Dinoflagellate species such as Karenia brevis, Karenia mikimotoi, Heterosigma akashiwo and Chatonella cf. antiqua are recognised toxic species implicated in various faunal mortalities. Toxic blooms of Karenia cristata were observed on the south coast of South Africa for the first time in 1988 and were responsible for mortalities of wild and farmed abalone. K. cristata and various other dinoflagellate species common along the South African coast, as well as K. mikimotoi (Isolation site: Norway, Univ. of Copenhagen) and K. brevis (Isolation site: Florida, BIGELOW), were tested for toxicity by means of a bioassay involving Artemia larvae as well as abalone larvae and spat. K. cristata, like K. brevis, contains an aerosol toxin; however, the toxin present in K. cristata has not yet been isolated and remains unknown. K. brevis was, therefore, used to determine which developmental phase of the bloom would affect abalone farms most, and whether ozone could be used as an effective mitigating agent. Of the 17 dinoflagellate species tested, K. cristata, Akashiwo sanguinea, K. mikimotoi and K. brevis pose the greatest threat to the abalone mariculture industry. K. brevis was most toxic during its exponential and stationary phases. Results suggest that ozone is an effective mitigation agent but its economic viability for use on abalone farms must still be investigated.     

OBSERVATION OF SAND-DWELLING TOXIC DINOFLAGELLATES (DINOPHYCEAE) FROM WIDELY DIFFERING SITES, INCLUDING TWO NEW SPECIES

Dinoflagellate associations, including toxic and potentially toxic benthic species, were examined in sand from South Water Cay and Carrie Bow Cay, Belize. The inshore sand habitat in localized areas of warm shallow lagoonal waters supported blooms of toxic assemblages of dinoflagellates. In the sand, the dominant microalgae were dinoflagellates; cyanobacteria were a minor component and diatoms were absent. Ciliates and nematodes were present. Assemblages of microorganisms in colored sand were examined for 4 consecutive days after which a storm washed away the patch. The sand-dwelling dinoflagellate assemblage included 16 species where densities ranged from as low as 1.3% to 15% of total cell densities. The dominant species was Scrippsiella subsalsa, having 1.8 × 10⁵ to 2.6 × 10⁵ cells g^-1 sand. Toxic dinoflagellates identified in the sand were Gambierdiscus toxicus, Ostreopsis lenticularis, Prorocentrum lima, Prorocentrum mexicanum, and Amphidinium carteri. The potentially toxic Ostreopsis labens, Gambierdiscus belizeanussp. nov., and Coolia tropicalis sp. nov. were also identified. Toxic and potentially toxic species represented 36% to 60% of total microalgal cell assemblage. The morphology of a new sand-dwelling species, Gambierdiscus belizeanus sp. nov., was examined with the scanning electron microscope. The plate formula was P_o, 3′, 7″, 6c, s?, 5?, 1p, and 2″″.Dimensions of G. belizeanus cells were 53–67 pm long, 54–63 μm wide, and 92–98 μm in dorsoventral depth. Cells were deeply areolated, ellipsoid in apical view, and compressed anteroposteriorly. The cells of G. belizeanus were identified by the cell's long, narrow, pentagonal, posterior intercalary plate (1p) wedged between the wide postcingular plates 2″’and 4″; 1p occupied 20% of the width of the hypotheca. The plate formula for Coolia tropicalis sp. nov. was P_o, 3′, 7″, 7c, 8s?, 5″″, and 2″″, Cell size ranges were 23–40 μm long, 25–39 μm wide, and 35–65 μm in dorsoventral diameter. Cells were spherical, smooth, and covered with scattered round pores. The epitheca was smaller than the hypotheca. Precingular plates 1″ and 7″ were small and narrow, and the first apical plate 1″ and precingular plate 6″ were the largest plates on the epitheca. The apical pore was straight and 7 μm long, and was situated in the apical plate complex. Cells of C. tropicalis were distinguished from C. monotis by the wedge-shaped plate 1′, a four-sided 3’plate, and a short apical pore.     

Development and application of LSU rRNA probes for Karenia brevis in the Gulf of Mexico, USA

The brevetoxin producing dinoflagellate, Karenia brevis, is the target of several monitoring and research programs in the Gulf of Mexico, where it forms extensive and frequently long-lived annual blooms that can cause human intoxication and fish kills, as well as severe economic losses to coastal communities. Rapid, reliable methods for the detection and enumeration of K. brevis cells, as well as their discrimination from morphologically similar species, are valuable tools for managers and scientists alike. Our aim was to produce a species-specific molecular probe that would serve as a tool to facilitate the efficient and reliable detection of K. brevis in the Gulf of Mexico. We sequenced a fragment of the large-subunit ribosomal RNA gene (LSU rDNA) from five K. brevis cultures isolated from the Texas Gulf coast, the Florida Gulf coast, and the Atlantic coast of Florida, and detected no differences among these isolates. A consensus sequence was thus compiled and compared to a previously published sequence from Karenia mikimotoi, the closest known phylogenetic relative to K. brevis, for the purpose of identifying unique K. brevis signature sequences. Fluorescently-labeled (FITC) oligonucleotide probes targeting these regions of the K. brevis LSU rRNA were designed to include at least two base pair differences, as compared to K. mikimotoi. Among seven probes designed, one uniquely identified all K. brevis isolates to the exclusion of all other species tested (Kbprobe-7), including a Gulf of Mexico K. mikimotoi isolate (Sarasota, FL) and several additional Gymnodinium species, as well as other dinoflagellate, diatom, and raphidophyte taxa. Importantly, K. brevis cells in samples taken during a 2001 bloom, fixed with a mixture of modified saline ethanol and 10% formalin, and stored at 4 °C for 7 months were successfully labeled with Kbprobe-7. In addition, preliminary analysis of labeled cells by flow cytometry revealed that K. brevis could be distinguished from K. mikimotoi in solution, suggesting other potential applications of this probe.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.     

NOVEL STEROLS OF THE TOXIC DINOFLAGELLATE KARENIA BREVIS (DINOPHYCEAE): A DEFENSIVE FUNCTION FOR UNUSUAL MARINE STEROLS?1

The “red tide” organism Karenia brevis (Davis) Hansen & Moestrup (=Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare Δ⁸⁽¹⁴⁾ sterols. The two predominant sterols, (24R)‐4α‐methylergosta‐8(14), 22‐dienol and (24R)‐4α‐methyl‐27‐norergosta‐8(14), 22‐dienol, were named gymnodinosterol and brevesterol and represent potentially useful biomarkers for K. brevis. A possible function for such unusual marine sterols is proposed whereby structural modifications render the sterols non‐nutritious to marine invertebrates, reducing predation and thereby enhancing the ability of the dinoflagellates to form massive blooms.     

Review of the cicada genus Karenia (Hemiptera: Cicadidae), with a description of one new species trapped by clapping hands and its entomogenous fungus

Abstract

The Oriental cicadas of the genus Karenia are reviewed and a key to the species is presented. One new species, K. chama Wei & Zhang, sp. nov., trapped by hand clapping and bamboo‐stick clicking, is described and illustrated. Information on its biology, as well as a few notes on its special sound‐production mechanism, is provided in some detail. The sound communication and mimicry of attracting cicadas, the associated cicada fungus, Elaphocordyceps sp., and the distribution of Karenia species are discussed.