DIEL CHANGES IN THE COMPOSITION OF PHOTOSYNTHETIC PIGMENTS AND CELLULAR CARBON AND NITROGEN IN CHATTONELLA ANTIQUA (RAPHIDOPHYCEAE)1

An axenic clonal culture of Chattonella antiqua (Hada) Ono was grown on a 12: 12 h LD cycle in a laboratory culture tank containing 1 m³ of f/2 medium. Diel changes in mean cell volume, cellular carbon (carbon content per cell), C/N ratio, cellular Chl a, Chl a/c ratio and carotenoid composition were observed. Mean cell volume and cellular C, N and pigments increased during the light period as a result of photosynthesis and decreased with increase of cell concentration by phased cell division during the dark period. These changes indicated that carbon assimilation and pigment synthesis occurred together during the light period. However, the patterns of increase were not the same since different diel patterns were also found in the ratios of C/N and chl a/c. Photosynthetic pigments were analyzed by reversed-phase high-performance liquid chromatography with ion-pairing solution. This analysis showed that the dominant carotenoids in C. antiqua were fucoxanthin, violaxanthin and β-carotene. Diel patterns of Chls a and c were similar to that of fucoxanthin but different from those of violaxanthin and β-carotene. The cellular contents of Chl a, fucoxanthin and carbon increased in a parallel manner during the light period. On the other hand, the increase of violaxanthin was restricted to only a few hours at the beginning of the light period during cell division cycles.     

Strain variability in fatty acid composition of Chattonella marina (Raphidophyceae) and its relation to differing ichthyotoxicity toward rainbow trout gill cells

Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C. marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4–29.4%) compared to intact cells (8.5–25.3%). In general, Japanese N‐118 C. marina was the highest producer of EPA (14.3–29.4%), and Mexican CMCV‐1 the lowest producer (7.9–27.1%). Algal cultures, free fatty acids from C. marina, and the two aldehydes 2E,4E‐decadienal and 2E,4E‐heptadienal (suspected fatty acid‐derived products) were tested against the rainbow trout fish gill cell line RTgill‐W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C. marina showed a potent toxicity toward gill cells (median lethal concentration, LC₅₀ (at 1 h) of 0.44 μg · mL^?1 in light conditions, with a complete loss of viability at >3.2 μg · mL^?1). Live cultures of Mexican C. marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E‐decadienal and 2E,4E‐heptadienal also showed high impact on gill cell viability, with LC₅₀ (at 1 h) of 0.34 and 0.36 μg · mL^?1, respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N‐118 and Mexican CMCV‐1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5 pmol · cell^?1 · hr^?1 for CMPL01, respectively). Our results indicate that C. marina is more ichthyotoxic after cell disruption and when switching from dark to light conditions, possibly associated with a higher production of superoxide anion and EPA, which may be quickly oxidized to produce more toxic derivates, such as aldehydes.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Life cycle of Chattonella marina (Raphidophyceae) inferred from analysis of microsatellite marker genotypes

Red tides of Chattonella spp. have caused continuous damage to Japanese aquaculture, however, the life cycle of this organism remains incompletely understood. To further investigate this matter, we assessed genotypes at 14 microsatellite markers in three varieties of Chattonella marina, viz., C. marina var. antiqua, C. marina var. marina, and C. marina var. ovata, to establish whether Chattonella undergoes asexual diploidization or sexual reproduction. After genotyping 287 strains of C. marina, all but one of these strains was shown to be heterozygous for at least some loci, and thus, in the diploid state, suggesting that Chattonella strains undergo sexual reproduction. In addition, we performed single‐cell amplification on ‘small cells’ that are derived from vegetative cells under dark and low‐nutrient conditions. The results indicated the existence of two types of small cells. The ‘Small cell Type 1’ was found to be heterozygous, genotypically equivalent to the vegetative cells, and is therefore diploid. These small cells may change to resting cells (cysts) directly. The ‘Small cell Type 2’ was homozygous at all analyzed loci, suggesting that these small cells are haploid and may be derived by meiosis. As fusion between small cells has previously been observed, the ‘Small cell Type 2’ may be the gamete of Chattonella. We present a construct of the full life cycle of Chattonella marina based on our own and previous results.     

The association of algicidal bacteria and raphidophyte blooms in South Carolina brackish detention ponds

Over the past 5 years, raphidophyte blooms have been frequently observed along the South Carolina coastal zone. During the 2002, 2003, and 2004 sampling seasons, we investigated temporal fluctuations of algicidal bacteria abundance against raphidophycean flagellates (Heterosigma akashiwo, Chattonella subsalsa, and Fibrocapsa japonica) using the microplate most probable number (MPN) method in three Kiawah Island brackish stormwater detention ponds (K1, K2, and K75). Local axenic isolates of H. akashiwo, C. subsalsa, and F. japonica were obtained and their susceptibility to algicidal bacteria tested. A total of 195 algicidal bacterial strains were isolated from raphidophyte blooms in the study ponds, and 6 of them were identified at the genus level, and the taxonomic specificity of their algicidal activity was tested against local (pond) and nonlocal isolates of raphidophytes (3 species, 10 total strains). In the ponds, a consistent association was found between raphidophyte bloom development and an increase in bacteria algicidal to the bloom species. In 12 of 15 cases, bloom decline followed the increase in algicidal bacteria to maximum abundances. Although variability was found in the taxonomic specificity of the algicidal bacteria effect (i.e. the number of raphidophyte species affected by a particular bacteria strain) and raphidophyte susceptibility (i.e. the number bacteria strains affecting a particular raphidophyte species), a toxic effect was always found when strains of a raphidophyte species were exposed to algicidal bacteria isolated from a bloom caused by that same species. The results suggest that algicidal bacteria may be an important limiting factor in raphidophyte bloom sustenance and can promote bloom decline in brackish lagoonal eutrophic estuaries.     

High toxicity of the novel bloom-forming species Chattonella ovata (Raphidophyceae) to cultured fish

A toxicological study of an axenic cell line of novel species Chattonella ovata Y. Hara et Chihara (Raphidophyceae) revealed that cultured species of sea bream (Pagrus major), horse mackerel (Trachurus japonicus), and yellowtail (Seriola quinqueradiata) were killed by 4.1–6.8 × 10³, 5.4 × 10³, and 2.8 × 10³ cells/mL, respectively. The sensitivity of the gill lamellae to C. ovata differed among the fish species tested. This finding revealed that C. ovata was highly toxic to the cultured fish. Histological examination showed that edema and hyperplasia of the secondary gill lamellae of red sea bream and horse mackerel occurred when exposed to, or killed by C. ovata, whereas severe damage in the gill lamellae was not observed in yellowtail. Chattonella produced high amounts of superoxide anion radicals and hydrogen peroxide, possibly responsible for the fish death observed. Based on the results of this study and occurrence of a red tide by this organism in China in 2001, we consider this organism to be one of the harmful algae in coastal waters. This is the first report demonstrating that C. ovata is highly toxic to fish, and that it produces superoxide and hydrogen peroxide.     

Verrucophora farcimen gen. et sp. nov. (Dictyochophyceae,Heterokonta)—a bloom‐forming ichthyotoxic flagellate from the Skagerrak,Norway1

Since 1998, a heterokont flagellate initially named Chattonella aff. verruculosa has formed recurrent extensive blooms in the North Sea and the Skagerrak, causing fish mortalities. Cells were isolated from the 2001 bloom off the south coast of Norway, and monoalgal cultures were established and compared with the Chattonella verruculosa Y. Hara et Chihara reference strain NIES 670 from Japan. The cells in Norwegian cultured isolates were very variable in size and form, being large oblong (up to 34 μm long) to small rounded (5–9 μm in diameter) with two unequal flagella, numerous chloroplasts, and mucocysts. The SSU and partial LSU rDNA sequences of strains from Norway and Japan were compared and differed by 0.4% (SSU) and 1.3% (LSU), respectively. Five strains from Norway were identical in the LSU rDNA region. Phylogenetic analyses based on heterokont SSU and concatenated SSU + LSU rDNA sequences placed C. aff. verruculosa and the Japanese C. verruculosa within the clade of Dictyochophyceae, with the picoflagellate Florenciella parvula Eikrem as the closest relative. Ultrastructure, morphology, and pigment composition supported this affinity. We propose the name Verrucophora farcimen sp. et gen. nov. for this flagellate and systematically place it within the class Dictyochophyceae. Our studies also show that C. verruculosa from Japan is genetically and morphologically different but closely related to V. farcimen. The species is transferred from the class Raphidophyceae to the class Dictyochophyceae and renamed Verrucophora verruculosa. We propose a new order, Florenciellales, to accommodate V. farcimen, V. verruculosa, and F. parvula.     

Phosphate metabolism during diel vertical migration in the raphidophycean alga, Chattonella antiqua

The ecological advantage of diel vertical migration on the nutrition and accumulation of Chattonella antiqua, which is one of the dominant red-tide forming phytoplankton species in the Seto Inland Sea of Japan, was examined using a large axenic culture tank, in which vertical stratification of salinity, temperature and nutrients was maintained, analogous to natural conditions observed when red tides occur. C. antiqua was capable of migrating through very sharp salinity and temperature gradients. At night the species migrated to the deep nutrient-rich water and assimilated nutrients. During the daytime it migrated to the nutrient-depleted surface water and used the accumulated nutrients for photosynthesis. Nitrogen uptake was synchronized with phosphate uptake. ³¹P-NMR spectroscopy during the migration experiment revealed that C. antiqua has the capability of nocturnal phosphate uptake in the deep nutrient-rich water, but no capability of synthesizing polyphosphate, which was considered to be the intracellular phosphate pool. These findings were compared with those reported for another raphidophycean, Heterosigma akashiwo. Although both species carry out vertical migration and nocturnal nutrient uptake, only H. akashiwo has the capability of making an intracellular polyphosphate pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

大亚湾春季卡盾藻种群动态及其赤潮成因分析

研究了2000年春季大亚湾古老卡盾藻/海洋卡盾藻(Chattonella antique/marina)种群动态及其与环境因子的关系。卡盾藻是一种沿岸分布的有害赤潮针孢藻,2000年春在大亚湾海域高密度高频率出现,最高细胞密度为129cells/mL。大亚湾合适的水温、盐度及丰富的营养盐含量为卡盾藻的生长提供了良好的环境条件,而较低的Si∶N及持续的阴雨天气抑制了小型硅藻的生长,使卡盾藻在浮游植物种群竞争中占据一定地位。孢囊萌发被认为是水体中卡盾藻营养细胞及其赤潮发生的种源,但孢囊需经过4个月以上、11℃以下低温处理后方可成熟和萌发,大亚湾冬季水温,不能满足卡盾藻孢囊休眠成熟的需要。作者推测,2000年春大亚湾高密度卡盾藻营养细胞的种源可能来源于由船舶压舱水所带来的成熟孢囊。       

海洋卡盾藻(香港株)溶血毒素的提取和分离

海洋卡盾藻(Chattonella marina)是我国南方沿海主要的鱼毒性赤潮生物,近年来由该藻形成的赤成已造成多起近岸养殖鱼类大量死亡的事件。为了弄清该藻毒素的基本成分特征,本文研究了室内培养条件下海洋卡盾藻(香港株)溶血毒素的提取方法,观察了溶血毒素对红细胞的溶血过程,采用薄层色谱法对溶血成分进行了初步分析。结果表明:海洋卡盾藻藻细胞的超声波破碎最适条件为功率400W,4℃下处理15min;通过显微镜观察,证实提取的毒素对血红细胞膜具破坏作用;海洋卡盾藻合成的溶血毒素至少含有4种组分,其中1种可能为为脂类,3种为糖脂类。该研究成果有助于我国今后进一步开展鱼毒性赤潮生物毒素的研究。