Cell cycle and DNA synthesis in a marine dinoflagellateAmphidinium carterae

Summary The incorporation of tritiated thymidine into the DNA ofAmphidinium carterae in synchronized cultures has been analyzed by nucleic acid separation and CsCl centrifugation. The phases of the cell cycle are well defined In spite of an unusual characteristic: superimposed on the S phase, synchronous for nuclear and plastidal DNA's, a residual incorporation is observed whenever a labelling pulse is given. The corresponding continuous DNA replication is insensitive to ethidium bromide.Abbreviations BET ethidium bromide - CPM radioactive counts per minute - HAP hydroxylapatite - J₁, J₂, ..., J₉: 1st 2d, ... 9th day of a culture - L, D Light, Dark periods - mM milli-moles - pM picomoles - MF macromolecular fractionation - PB phosphate buffer - SSC standard saline citrate (0.15 M NaCl, 0.015 M Na citrate, pH 7).     

Identification of very-long-chain polyunsaturated fatty acids from Amphidinium carterae by atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy

A method is described for the enrichment of very-long-chain polyunsaturated fatty acids (VLCPUFAs) from total fatty acids of Amphidinium carterae and their identification as picolinyl esters by means of microbore liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The combination of argentation TLC and LC-MS/APCI was used to identify unusual VLCPUFAs up to hexatriacontaoctaenoic acid. Two acids, 36:7n-6 and 36:8n-3, were also synthesized to unambiguously confirm their structure. The possibilities of VLCPUFAs biosynthesis are proposed.     

Odd-numbered very-long-chain polyunsaturated fatty acids from the dinoflagellate Amphidinium carterae identified by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

A method is described for the enrichment of odd very-long-chain polyunsaturated fatty acids (VLCPUFAs) by means of RP-HPLC and argentation TLC from total fatty acids of the dinoflagellate A. carterae and their identification as picolinyl esters by means of microbore liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The combination of argentation TLC and LC-MS/APCI was used to identify rare and unusual odd VLCPUFAs up to nonacosahexaenoic acid. Two acids, (allZ)-nonacosa-11,14,17,20,23-pentaenoic acid (29:5n-6) and (allZ)-nonacosa-11,14,17,20,23,26-hexaenoic acid (29:6n-3), were synthesized for the first time to unambiguously confirm their structure. Possible biosynthetic pathways for odd VLCPUFAs are also proposed.     

海洋浮游生物介导的弧菌传播扩散机制研究进展

弧菌在全球范围内广泛存在,弧菌病的暴发和流行不仅给海水养殖业造成巨大的经济损失,还严重威胁人类健康。一些弧菌可以通过与环境中浮游植物和浮游动物的相互作用提高自身存活率和环境持久性,并进行跨海域传播。本文综述了海洋浮游生物作为部分弧菌物种储库的作用及影响因素,以及气候变化和人类活动等因素驱动下浮游生物间接介导的弧菌传播扩散特性;并重点介绍了弧菌特定的结构、代谢途径和次级代谢产物在其与浮游生物互作过程中发挥的独特作用及其机制;提出今后应从分子层面深入解析浮游生物与弧菌的相互作用机制,从全球尺度阐释浮游生物介导的弧菌跨海域传播机制,建立弧菌疾病传播预测模型和预警系统,为浮游生物介导的弧菌传播扩散风险的防控提供重要信息。     

The relationship between calcification and photosynthesis in the coccolithophorid Pleurochrysis carterae

Coccolithophorids are one of the dominant groups of marine phytoplankton. They are found in large numbers throughout the surface euphotic zone of the ocean, and are able to form large-scale blooms that persist for long periods of time. Coccolithophorid cells are covered by species-specific calcium carbonate crystals of various structures. In the process of calcification in coccolithophorids, Ca²⁺ is absorbed into cells from the culture medium, and a coccolith unit is formed inside the cell. Then, the coccolith unit extrudes to the cell surface where it is constructed into crystal layers. The formation of these crystals is regulated by cellular metabolism under different environmental conditions. The carbon biogeochemical cycle in the coccolithophorids involves both photosynthetic and calcification processes, which not only play an important role in population dynamics, but also in the global carbon cycle and climate change. However, one important question remains, namely, whether the relationship between photosynthesis and calcification is species-dependent. Previous studies have yielded controversial results, even in the same species. In this paper, we selected Pleurochrysis carterae, a coccolithophore species that frequently blooms in coastal areas, to study the relationship between calcification and photosynthesis. First, we studied population growth in a batch culture over several days. For batch cultures, P. carterae was inoculated into a 10 L bioreactor at an initial cell density of approximately 5 × 10⁴ cells mL^-1. The culture conditions were optimal for cell growth. Dissolved oxygen (DO) was detected during all the culture period, and the rate of photosynthetic oxygen evolution was calculated according the DO changes during the 12-h illumination period. Algal samples (10 mL) were collected during the population growth phases. The calcium carbonate content on the cell surface was determined each day by chemical titration. Next, we studied the relationship between photosynthesis and calcification at the cellular level by observing patterns of recalcification during a 12-h period. In this study, non-calcified cells were obtained by decalcifying calcified cells collected during the exponential growth period in MES-NaOH buffer solution (pH 5.5). The non-calcified cells were inoculated into culture media containing different concentrations of Ca²⁺ (0, 5, 20, 40, 50, or 100 mg L^-1). The rate of recalcification was determined by microscopic analyses in which the number of recalcified cells per 100 cells was counted at 0, 3, 6, 9, and 12 h of culture. Ca²⁺ absorbed into the cell was detected by measuring the fluorescence intensity of Fluo-3/AM labeled Ca²⁺. The rate of photosynthetic oxygen evolution in the non-calcified cell cultures was detected by measuring the changes in dissolved oxygen during the 12-h illumination period. The results showed that during the population growth period, the rate of photosynthetic oxygen evolution was inversely related to the calcium carbonate content per cell. When the amount of calcium carbonate on the cell surface increased, the relative photosynthetic ability (the rate of photosynthetic oxygen evolution) decreased, and vice versa. Both recalcification rates and photosynthetic oxygen evolution were affected by the extracellular calcium concentration. Non-calcified cells showed different recalcification abilities at different extracellular Ca²⁺ concentrations. The recalcification rate of non-calcified cells was positively correlated with the extracellular calcium concentration when [Ca²⁺] in the medium ranged from 0 to 100 mg L^-1. However, photosynthetic oxygen evolution was suppressed at higher cell calcification rates, especially when extracellular [Ca²⁺] was 50–100 mg L^-1. Our analyses of the population growth process and the cell recalcification process confirmed that photosynthesis is inversely related to calcification in P. carterae.     

Occurrence and toxicity of Amphidinium carterae Hulburt in the North Arabian Sea

The occurrence and toxicity of Amphidinium carterae Hulburt is hereby reported for the first time from the North Arabian Sea on the coast of Pakistan. The concentrations of 1.2 × 10⁴ cells ml⁻¹ were found in intertidal pools that were also inhabited by the brown macroalga Sargassum wightii. Both wild and cultured A. carterae cells were tested for ciguatera toxicity through exposure to brine shrimp nauplii (Artemia salina) and albino mice. Although the brine shrimp did not appear to be affected mortalities in mice ranged between 13 and 16% at doses of 7.2 × 10⁴ and 2.5 × 10⁵ cells ml⁻¹, respectively. When mice were affected pharmacological effects such as muscle contraction in lower back area, increased respiration, immobility and paralysis in hind limbs were observed for 2 h. These effects appeared to be reversible and gradually disappeared within 24 h.     

Evaluation of the mutagenic properties of the coccolithophorePleurochrysis carterae (Haptophyceae) as a potential human food supplement

The mutagenicity of the algaPleurochrysis carterae for use as human food was tested by the Ames method with the modification of pre-incubation, by usingSalmonella typhimurium TA98, TA100, TA1535, TA1537 andEscherichia coli WP2uvrA. The freeze-dried powder ofP. carterae was not mutagenic to any strain either with or without S9 mix. In view of the absence of adverse effects ofP. carterae in this mutagenicity study, it is suggested thatP. carterae is safe for human consumption as a human food supplement.Author for correspondence     

Identification and survival of the causal organism of leaf smut disease of cowpea in Nigeria

Protomycopsis phaseoli (Ramak and Subram) is the causal agent of the cowpea leaf smut disease in Nigeria and not Entyloma vignae as claimed by some authors. This pathogen formed dark ash-grey to sooty-black lesions of 3–10 mm in diameter, while young lesions had yellow haloes. P. phaseoli produced dark reddish-brown chlamydospores that are globose to oval measured 23.8 μm, thick-walled and rugose. The chlamydospores germinated and produced globose vesicles. The pathogen grew on potato dextrose agar only when the leaf tissue was dipped in acidified water (1% H₂SO₄). The organism was slowly growing at 24–28 °C with snow white colour. Chlamydospores of P. phaseoli in infected cowpea leaves survived longer when buried in the soil for five months than when they were left on the soil surface for the same period at temperatures (26–27 °C) and humidity (70–82%) prevailing in Ibadan. Destruction of leaf debris before crop emergence, long period of rotation and no tillage cropping are suggested to prevent the onset and spread of leaf smut disease of cowpea. This revised version was published online in June 2006 with corrections to the Cover Date.     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

The invariance of macromolecular composition with altered light limited growth rate of Amphidinium carteri (dinophyceae)

The effect of irradiance on the growth rate, macromolecular composition and photosynthetic carbon metabolism of Amphidinium carteri was studied in batch culture. Growth rate increased linearly with increasing irradiance up to a maximum growth rate of 0.04 h^-1 at an irradiance of 80 Em^-2s^-1. In contrast to a number of other studies on both prokaryotic and eukaryotic microorganisms, ours showed that cellular content of RNA, DNA, protein and carbohydrate of A. carteri were invariant with growth rate over the range =0.04 to 0.007 h^-1. The invariant macromolecular composition was correlated with a constant modal cell volume. Chlorophyll and lipid per cell increased with decreasing irradiance. The distribution of [¹⁴C]-bicarbonate in the major end products of photosynthesis after incubation with isotope for 14% of a doubling time showed that the percentage carbon in the chloroform (lipids and pigments) fraction increased with decreasing irradiance while that of the trichloroacetic acid soluble (carbohydrate) fractions decreased. The percentage of isotope in the trichloroacetic acid insoluble (protein) fraction and methanol: water fraction (metabolites) remained constant. Because this species, under light-limited growth, differs from other organisms so far studied, more species must be similarly examined before nucleic acid content is used as an index growth rate in the field.This paper is presented with our best wishes on the occasion of Professor G. Drews 60th birthday     

The mitotic apparatus in the dinoflagellateAmphidinium carterae

Summary Aspects of mitosis in the dinoflagellateAmphidinium carterae have been examined using TEM, SEM and fluorescence immunochemistry. The extranuclear spindle is composed of 2–4 bundles of microtubules arranged into two interdigitated half-spindles. Unlike previous reports of dinomitosis, the spindle bundles converge at the poles. These bundles of microtubules are inserted into a multilobed, vesiculate body containing electron opaque, amorphous material. This spindle pole body has ribosomes associated with it and is continuous with the endoplasmic reticulum. Chromosomes are attached to the nuclear envelope, which is persistent throughout mitosis. Kinetochore microtubules attach to the nuclear envelope via elongate electron dense kinetochores (one microtubule per daughter kinetochore). Several microtubules pass alongside the kinetochore, forming a halo of 3–4 spindle microtubules. Electron dense connections can be seen between some of these microtubules and the kinetochore. Chromosome segregation appears to be a function of spindle elongation (anaphase B), since chromosome-to-pole distance (anaphase A) remains relatively unchanged throughout mitosis.Abbreviations DABCO 1,4 diazabicyclo(2,2,2)octane - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,NN-tetraacetic acid - PIPES piperazine-N,N-bis(2-thanesulfonic acid) Supported by a Charles and Johanna Bush Predoctoral Fellowship to S. B. B.     

Comparative morphology and molecular phylogeny of Apicoporus n. Gen.: a new genus of marine benthic dinoflagellates formerly classified within Amphidinium

The composition of the dinoflagellate genus Amphidinium is currently polyphyletic and includes several species in need of re-evaluation using modern morphological and phylogenetic methods. We investigated a broad range of uncultured morphotypes extracted from marine sediments in the Eastern Pacific Ocean that were similar in morphology to Amphidinium glabrum Hoppenrath and Okolodkov. To determine the number of distinct species associated with this phenotypic diversity, we collected LM, SEM, TEM and small subunit ribosomal DNA sequence information from different morphotypes, including the previously described A. glabrum. Both comparative morphological and molecular phylogenetic data supported the establishment of a new genus, Apicoporus n. gen., including at least two species, A. glaber n. comb., and A. parvidiaboli n. sp. Apicoporus is characterized by having amphiesmal pores and an apical pore covered by a hook-like protrusion; neither of these characters has been observed in other athecate dinoflagellates. The posterior end of Apicoporus parvidiaboli possessed varying degrees of "horn formation", ranging from slight to prominent. By contrast, the posterior end of Apicoporus glaber was distinctively rounded and lacked evidence of horn formation. Although these species were previously interpreted to be obligate heterotrophs, TEM and epifluorescence microscopy demonstrated that some cells of both species had unusually small but otherwise typical dinoflagellate plastids. The number and density of plastids in any particular cell varied significantly in the genus, but the plastids were almost always concentrated at the posterior end of the cells or around the nucleus. The presence of cryptic photosynthetic plastids in these benthic species suggests that photosynthesis might be much more widespread in dinoflagellates than is currently assumed.     

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.     

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

不同光照条件下米氏凯伦藻和东海原甲藻生长的温度生态幅

米氏凯伦藻和东海原甲藻是我国东南沿海地区赤潮的主要优势种。为定量获取米氏凯伦藻和东海原甲藻生长的温度生态幅,根据3个光照水平(28.32,75.06,111.66μmol m~(-2)s~(-1))条件下4个温度水平(18,22,25,28℃)对米氏凯伦藻和东海原甲藻生长特性的室内培养实验结果,并结合Shelford耐受性定律建立了基于温度的米氏凯伦藻和东海原甲藻比生长率的耐受性模型,最后根据前期的研究成果分别获取了米氏凯伦藻和东海原甲藻生长的最适温度、适温范围及耐受温度范围。结果表明,无论是米氏凯伦藻还是东海原甲藻,在相同培养光照条件下,在设定的温度水平范围内,分别存在一个适宜米氏凯伦藻和东海原甲藻的最适生长温度T_(opt),且当T≤T_(opt)时,米氏凯伦藻和东海原甲藻细胞密度和比生长率随着温度的升高而显著增大;而当T≥T_(opt)时,米氏凯伦藻和东海原甲藻细胞密度和比生长率随着温度的升高而显著减小。随着培养光照强度的升高,米氏凯伦藻和东海原甲藻细胞密度和比生长率均呈现"先升后降"的变化趋势。建立的藻类生长温度耐受性模型与谢尔福德耐受定律较为吻合,定量获取米氏凯伦藻在3个光照水平(28.32,75.06,111.66μmol m~(-2)s~(-1))下的最适生长温度分别为22.48,22.37,22.33℃;适温范围分别为17.93—27.03,17.82—26.92,17.78—26.88℃;耐受温度范围分别为13.38—31.58,13.27—31.47,13.23—31.43℃;东海原甲藻在3个光照水平(28.32,75.06,111.66μmol m~(-2)s~(-1))下的最适生长温度分别为22.10,21.99,21.93℃;适温范围分别为17.59—26.61,17.48—26.5,17.42—26.44℃;耐受温度范围分别为13.08—31.12,12.97—31.01,12.91—30.95℃。     

Uptake of sulphate,taurine, cysteine and methionine by symbiotic and free-living dinoflagellates

Sulphate uptake by Amphidinium carterae, Amphidinium klebsii and Gymnodinium microadriaticum grown on artificial seawater medium with sulphate, cysteine, methionine or taurine as sulphur source occurred via an active transport system which conformed to Michaelis-Menten type saturation kinetics. Values for K _m ranged from 0.18–2.13 mM and V _max ranged from 0.2–24.2 nmol · 10⁵ cells^–1 · h^–1. K _m for symbiotic G. microadriaticum was 0.48 mM and V _max was 0.2 nmol · 10⁵ cells^–1 · h^–1. Sulphate uptake was slightly inhibited by chromate and selenate, but not by tungstate, molybdate, sulphite or thiosulphate. Cysteine and methionine (0.1 mM), but not taurine, inhibited sulphate uptake by symbiotic G. microadriaticum, but not by the two species of Amphidinium. Uptake was inhibited 45–97% under both light and dark conditions by carbonylcyanide 3-chlorophenylhydrazone (CCCP); under dark conditions sulphate uptake was 40–60% of that observed under light conditions and was little affected by 3-(3,4-dichlorophenyl) 1,1-dimethylurea (DCMU).The uptake of taurine, cysteine and methionine by A. carterae, A. klebsii, cultured and symbiotic G. microadriaticum conformed to Michaelis-Menten type saturation kinetics. K _m values of taurine uptake ranged from 1.9–10 mM; for cysteine uptake from 0.6–3.2 mM and methionine from 0.001–0.021 mM. Cysteine induced a taurine uptake system with a K _m of 0.3–0.7 mM. Cysteine and methionine uptake by all organisms was largely unaffected by darkness or by DCMU in light or darkness. CCCP significantly inhibited uptake of these amino acids. Thus energy for cysteine and methionine uptake was supplied mainly by respiration. Taurine uptake by A. carterae was independent of light but was inhibited by CCCP, whereas uptake by A. klebsii and symbiotic G. microadriaticum was partially dependent on photosynthetic energy. Taurine uptake by cultured G. microadriaticum was more dependent on photosynthetic energy and was more sensitive to CCCP. Cysteine inhibited uptake of methionine and taurine by cultured and symbiotic G. microadriaticum to a greater extent than in the Amphidinium species. Methionine did not greatly affect taurine uptake, but did inhibit cysteine uptake. Taurine did not affect the uptake of cysteine or methionine.     

Serial reconstruction of the mitochondrial reticulum in the coccolithophorid,Pleurochrysis carterae (Prymnesiophyceae)

Summary A serial reconstruction of the chondriome ofPleurochrysis carterae (Braarudet Fagerland) Christensen has revealed a single, reticulated mitochondrion branching throughout the cell. The occurrence of a single mitochondrion in unicellular algae is briefly reviewed and its phylogenetic significance is discussed.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.