Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Microscopic eukaryotes living in a dying lake (Lake Koronia, Greece)

The morphological and phylogenetic diversity of the microscopic eukaryotes of the Lake Koronia water column was investigated during a mass kill of birds and fish in August–September 2004. The dominant morphospecies corresponded to the known toxin-producing species Prymnesium parvum , followed by Amoebidium sp., a taxon belonging to the group of parasitic Mesomycetozoea , and the common chlorophyte Pediastrum boryanum. Prymnesium exhibited heteromorphic life-cycle stages (flagellate and nonmotile coccoid cells). Phylogenetic analysis with 18S rRNA gene suggested that these heteromorphic stages belonged to the Platychrysis – Prymnesium monophyletic group. The most abundant phylotype was almost identical to P. boryanum . The fungal phylotypes were related to the Chytridiomycota , and the ciliate-like ones were closely related to Enchelys polynucleata and Pattersoniella vitiphila . Two phylotypes representing novel members belonging to the Jakobida and the Apicomplexa were also found. The microscopic eukaryotes of Lake Koronia include several organisms that are related to parasitic life modes.     

舞三毛金藻的分类和生物学

采用光镜和电镜对引起我国养殖鱼类中毒并造成大批死亡的三毛金藻进行了详细观察,系统地研究了其特殊运动方式,形态结构,繁殖方法,孢囊,鳞片形式及纹饰,两种鞭毛的结构和功能,并对其生物学等也进行了调查研究,证明在天津地区引起鱼类中毒的是舞三毛金藻(Prymnesium saltans Massart et Conrad,1926)。同时认为鳞片结构可作为属的特征之一,但不能作为种的唯一特征。本文对舞三毛金藻亚显微结构及孢囊形态描述,进一步补充和完善了种的鉴定特征。     

Suitability of Selected Marine Algae for Growing the Marine Heterotrich Ciliate Fabrea salina1

ABSTRACT. Forty-five axenically grown algal (sensu lato) species representing six divisions—that is. 13 Chlorophyceae, 14 Chrysophycophyta, five Dinophycophyta, seven Cryptophycophyta, two Rhodophycophyta, and four Cyanochloronta—were aseplicaily presented separately as potential food sources to the marine helerotrich ciliate Fabrea salina under standardized algal number, medium, lighting, and temperature. The algae can be placed into three groups based on their effect on the intrinsic growth rate of the ciliate. Nutritious: Rhodomonas lens, cryptomonad LIS₁, Dunaliella parva, Prasinodadus marinus, Chroomonas salina, D. tertiolecta, Chaeloceros galvestonensis, D. primolecta, Phaeodactylum tricornutum, D. salina, Isochrysis galbana, Cylindrothecaclosterium, cryptomonad strains M₂, WH₂ & FSA, Chroomonas sp., P. lubricus, and Peridinium trochoideum. Maintamers: Cyanobacterium strain Tigriopus blue green, P. triquetum. Monochrysis lutheri, Exuviella gracilis, Platymonas tetrathele. Cyclotella caspa, Crypthecodinium cohnii, Prasinocladus C₅ strain, D. viridis, Nannochloris occulata, Tetraselmis gracilis, Anacystis marinum, Rhodosorus marinum, and Thalassiosira pseudonana. Nonnutritious: Stichococcus immobilis, Hymenomonas sp. strain 150, Syracosphaera sp. strain 181, Tetraselmis verrucosa, Thalassiosira fluviatilis, Microcoleus chthonoplastes, Synechococcus sp., Pavlova gyrans, Prymnesium parvum, Coccolithus huxleyi, Olisthodiscus luteus, Amphidinium carterii, and Porphyridium aerugineum. There was no apparent relationship between a given taxon and the nutritional value of the group, with the possible exception of the Cryptophycophyta.     

On the allelochemical potency of the marine dinoflagellate Alexandrium ostenfeldii against heterotrophic and autotrophic protists

Three strains of the marine dinoflagellate Alexandrium ostenfeldiiof different geographic origin were tested for their short-termdeleterious effects on a diversity of marine protists. All A.ostenfeldii strains were capable of eliciting an apparent allelochemicalresponse, but the various protistan target species were differentiallyaffected. Protists that were negatively affected by exposureto cells of A. ostenfeldii and associated extracellular metabolitescomprised both autotrophs (Rhodomonas sp., Dunaliella salina,Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina,Amphidinium crassum, Rimostrombidium caudatum). Observed effectsincluded immobilisation (e.g. of O. marina), morphological changes(e.g. in D. salina) and/or aberrant behaviour (e.g. of R. caudatum),mainly as preliminary stages of cell lysis. Immobilization andlytic effects against O. marina were strongly dependent on A.ostenfeldii cell concentrations. Effects also differed substantiallyamong strains and different batch cultures of the same strain.Values of EC₅₀, defined as the A. ostenfeldii cell concentrationcausing lysis of 50% of O. marina cells, ranged from 0.3 to1.9 x 10³ mL^–1, depending on the A. ostenfeldii strain.The autotrophic dinoflagellate Scrippsiella trochoidea reactedto exposure to A. ostenfeldii cells by formation of temporary(ecdysal) cysts, whereas, in contrast, the flagellates Emilianiahuxleyi and Prymnesium parvum and the ciliate Strombidium sp.were relatively refractory or even unaffected. As long as cellsdid not lyse, the fluorescence yield of target autotrophs, estimatedby pulse-amplitude modulation fluorometry, did not significantlychange during the first 3 h of incubation, suggesting that allelochemicalsproduced by A. ostenfeldii caused no short-term negative effectson the photosynthetic apparatus. Overall, the allelochemicalresponses of target species showed no obvious relationship tocell quota or extracellular concentrations of either toxic macrocyclicimines (spirolides) or tetrahydropurine neurotoxins (saxitoxinand analogues) produced by various strains of A. ostenfeldii.Instead, the potency of A. ostenfeldii, eliciting immobilizationand lytic species-specific responses in potential predatorsand competitors, is consistent with the existence of an allelochemicalmechanism unrelated to the bioactivity of known phycotoxinsof the genus Alexandrium.     

COMPARATIVE STUDIES ON ALDOLASE ACTIVITY IN MARINE PLANKTONIC ALGAE,AND THEIR EVOLUTIONARY SIGNIFICANCE1

Fructose diphosphate aldolase activity was examined in acetone powders and cell-free extracts of 15 photoautotrophically grown marine planktonic species belonging to 6 algal divisions as follows: Chlorophyta: Tetraselmis maculata, Dunaliella tertiolecta; Chrysophyta: Monochrysis lutheri, Isochrysis galbana, Prymnesium parvum, Coccolithus huxleyi; Bacillariophyta: Phaeodactylum tricornutum, Skeletonema costatum, Cyclotella nana; Cryptophyta: Cryptomonas sp., Rhodomonas lens, Hemiselmis virescens; Pyrrophyta: Amphidinium carteri; Cyanophyta: Anacystis marina, Agmenellum quadruplicatum. Indications of the types of aldolase (Rutter's classes) present in each alga were obtained from comparative studies of the effects of pH and of the following reagents on the activity: ethylenediamine tetraacetate, dithiothreitol, p-chloromercuriphenyl sulfonate. Type I (higher plant-animal type) aldolase only was indicated in the 2 chlorophytes, in I chrys-ophyte (M. lutheri), and in 1 bacillariophyte (P. tricornutum), while the remaining algae appeared to contain either exclusively or principally Type II (bacterial-fungal type) aldolase. The evolutionary implications of these findings are discussed.     

Enzymatic consumption of carbonyl sulfide (COS) by marine algae

We show that the marine algae Mantoniella squamata, Prymnesium parvum, and Amphidinium klebsii take up carbonyl sulfide (COS) from their surrounding medium. Inhibitor studies confirm that this COS uptake is catalyzed by the enzyme carbonic anhydrase, which was not detectable with conventional methods. As shown for M. squamata, the COS uptake can be dependent on the growth conditions. Furthermore, COS uptake shows a clear positive correlation with the COS concentration in the growth medium. The value of K_1/2 for the COS uptake was estimated to be around 222 mol/m³. The COS consumption by the marine algae species investigated was estimated to be negligible compared to the photoproduction and hydrolysis of COS in seawater.     

小三毛金藻对盐碱土池塘毒害鱼类的防治研究

小三毛金藻（PrymnesiumparvumCarter）是一种有害藻类。通过以硫酸盐为主的盐碱土池塘的对照试验,总结出采用不同药物、黄泥土、酸性化肥和追施有机肥、提高池塘肥力、促使其它藻类大量繁殖;同时不定期进行水质分析、加注新水、降低盐度、调节pH值等防治措施,使小三毛金藻得到有效的控制。其防治效率达95％以上。自1985年在陕西省大荔、合阳引用这种防治技术到1997年使万亩盐碱地池塘养鱼水面未受到小三毛金藻大的危害。     

Toxin-assisted micropredation: experimental evidence shows that contact micropredation rather than exotoxicity is the role of Prymnesium toxins

Blooms of Prymnesium parvum can severely harm fish and zooplankton, presumably through the release of allelopathic exotoxins that offer advantages for Prymnesium in its interactions with competitors and prey. We show that Prymnesium attaches to zooplankton and fish, causing mortality, whereas exposure of these organisms to Prymnesium across a permeable membrane does not cause mortality. We also show that Prymnesium exotoxins are released independently of contact toxicity only in response to experimental procedures or natural causes of stress. Our results are consistent with the idea that toxins have evolved for release during cell-to-cell contact in support of heterotrophy. The evolution of toxin-assisted micropredation would be consistent with mechanisms of natural selection favouring individual fitness as opposed to broadcast allelopathy from which the benefits are more dispersed. Research into the toxicity of Prymnesium and other harmful algal species may profit from focus on processes following physical contact with potential prey.     

Aspects of the ecology of Prymnesium parvum (Haptophyta) and water chemistry in the Norfolk Broads, England

SUMMARY. Prymnesium parvum , an ichthyotoxic phytoplankter, has been recorded, at times abundantly, in the River Thurne, Norfolk, and its associated Broads. Its occurrence has been apparently more frequent and its population sizes probably larger since the late 1960s than previously and fish mortalities due to it now occur almost annually.
The Thurne system is brackish and may have become more so, due to exploitation by drainage pumps, of a saline water table, in recent years. Evidence for this is conflicting, but in any case an increase in salinity is unlikely to have made increased Prymnesium growth more likely. Eutrophication of some Broads in the system is most likely to have increased the populations of Prymnesium since the late 1960s and data are presented on the present water chemistry of the system for comparison with previous records, and on current phytoplankton and Prymnesium crops in different parts of it.
P. parvum has been isolated from the system as a unialgal culture and compared in morphology, salinity tolerance and ichthyotoxicity with a strain of P. parvum from Israel. The Broads strain differs slightly in size and pigmentation, but not in salinity tolerance. In culture it produces more ichthyotoxin than the Israeli strain.     

Automated detection and enumeration for toxic algae by solid-phase cytometry and the introduction of a new probe for Prymnesium parvum (Haptophyta: Prymnesiophyceae)

Harmful algal blooms have a severe impact on aquaculture andfishery and can be caused by toxic haptophytes and dinoflagellates.Different toxic species, which are not easy to distinguish fromtheir morphologically similar and non-toxic relatives, occurin both groups. Sequencing of the large subunit ribosomal RNAof different strains and taxonomic relatives allowed the designof a probe specific to the toxic Prymnesium parvum spp. Forthe rapid detection and enumeration of Prymnesium and Alexandriumcells in cultures and environmental samples, respectively, protocolsfor fluorescence in situ hybridization were adapted for automateddetection by a solid-phase cytometer, the ChemScan. This cytometerenables the automated counting of fluorescently labelled cellson a membrane filter and subsequently a microscopic verificationof these results by the user, because the motorized stage ofthe microscope is driven to each positive signal by the computersoftware to localize that cell on the filter. With this fastdetection method, it was possible to detect, enumerate and verifymicroalgal cells on a filter, with a detection limit of onecell per membrane filter.     

Changes in pigmentation of phytoplankton species during growth and stationary phase — consequences for reliability of pigment-based methods of biomass determination

In applied water ecology several methods for estimating the biomass or activity of phytoplankton depend on the proportion of accessory pigments (xanthophylls) to chlorophyll a. Therefore, changes in pigmentation during growth and stationary phase were investigated in four different species (Amphidinium klebsii, Euglena gracilis, Prymnesium parvum, Cryptomonas ovata) typical representatives of the major algal groups. The ratios of the different xanthophylls to chlorophyll a depended not only on the growth phase, but also on the species. InAmphidinium andEuglena, the ratio of xanthophylls to chlorophyll rises continuously during the growth phase and declined during the stationary phase. InPrymnesium, quantitative pigmentation was found to be nearly independent of the growth phase. InCryptomonas, however, this ratio was relatively constant during growth, but increased in the stationary phase. In contrast to higher plants, in which the breakdown of chlorophylls occurs before that of the xanthophylls, in three of the species both pigment classes were reduced in parallel when the cultures were in the stationary phase. AgingCryptomonas, however, exhibited a pigment breakdown pattern similar to higher plants. The use of these findings for the widely applied biomass determination by chlorophyll fluorescence and for other pigment-based methods is discussed.     

海洋浮游藻类无机碳利用机理的研究

为了认识海洋浮游藻类在碳充足和碳受限条件下对水体中溶解无机碳(DIC)的利用方式与可能机理,对13种海洋浮游藻类在不同pH和CO2浓度及不同DIC条件下细胞外碳酸酐酶(CA)的活性进行了分析测定.结果显示:13种藻中,只有Amphidinium carterae和Prorocentrum minimum在碳充足条件下具细胞外CA活性.Melosira sp.、Phaeodactylum tricornutum、Skeletonema costatum、Thalassiosira rotula、Emiliania huxleyi和Pleurochrysis carterae则在碳受限条件下才具细胞外CA活性.Chaetoceros compressus、Glenodinium foliaceum、Coccolithus pelagicus、 Gephrocapsa oceanica和Heterosigma akashiwo即使在碳受限条件下也未检测到细胞外CA活性.应用封闭系统中pH漂移技术和阴离子交换抑制剂4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS)等的研究表明,Coc. pelagicus和G. oceanica可通过阴离子交换机制进行HCO-3的直接利用.H. akashiwo没有潜在的HCO-3直接利用或细胞外CA催化的HCO-3利用.     

Observations of the biology and parasites of postsmolt Atlantic salmon, Salmo salar, from the Norwegian sea

The main food items of postsmolt Atlantic salmon caught in the Norwegian sea during summer 1991 were Parathemisto spp. and herring ( Chipca harengus ). The most prevalent parasite species were Chloromyxion sp., Apatemon sp., H. adunction, A simplex and I. salmonis .     

AMPHIDINIUM REVISITED. II. RESOLVING SPECIES BOUNDARIES IN THE AMPHIDINIUM OPERCULATUM SPECIES COMPLEX (DINOPHYCEAE), INCLUDING THE DESCRIPTIONS OF AMPHIDINIUM TRULLA SP. NOV. AND AMPHIDINIUM GIBBOSUM. COMB. NOV.1

Amphidinium operculatum Claparède et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.     

STEROLS OF SOME MARINE PRYMNESIOPHYCEAE

Sterols were identified in six marine prymnesiophyte isolates, some of which appear to have value as bivalve food. The principal sterol in Pleurochrysis carterae (Milford #961) and an unidentified prymnesiophyte (CCMP1215) was 24-methylcholesta-5,22-dienol, a common sterol in prymnesiophytes. Isolates CCMP594, CCMP609, and CCMP459 contained either 24-ethylcholesta-5,22-dienol or 24-ethylcholest-22-enol as the major sterol. In addition, Pavlova pinguis (CCMP609) and Pavlova sp. (CCMP459) contained the unusual dihydroxysterols 24-methylpavlovol and 24-ethylpavlovol, which have been found only in members of the Pavlovales. Prymnesium parvum contained cholesterol without traces of other sterols. Compared to the other isolates, the quantity of sterols was extremely low in P. parvum.     

Intra- and interspecific competition among helminth parasites: effects on Coitocaecum parvum life history strategy, size and fecundity

Larval helminths often share intermediate hosts with other individuals of the same or different species. Competition for resources and/or conflicts over transmission routes are likely to influence both the association patterns between species and the life history strategies of each individual. Parasites sharing common intermediate hosts may have evolved ways to avoid or associate with other species depending on their definitive host. If not, individual parasites could develop alternative life history strategies in response to association with particular species. Three sympatric species of helminths exploit the amphipod Paracalliope fluviatilis as an intermediate host in New Zealand: the acanthocephalan Acanthocephalus galaxii, the trematode Microphallus sp. and the progenetic trematode Coitocaecum parvum. Adult A. galaxii and C. parvum are both fish parasites whereas Microphallus sp. infects birds. We found no association, either positive or negative, among the three parasite species. The effects of intra- and interspecific interactions were also measured in the trematode C. parvum. Both intra- and interspecific competition seemed to affect both the life history strategy and the size and fecundity of C. parvum. Firstly, the proportion of progenesis was higher in metacercariae sharing their host with Microphallus sp., the bird parasite, than in any other situation. Second, the intensity of intraspecific competition apparently constrained the ability of metacercariae to adopt progenesis and limited both the growth and egg production of progenetic individuals. These results show that the life history strategy adopted by a parasite may be influenced by other parasites sharing the same host.     

Bioprospecting for oil producing microalgal strains: evaluation of oil and biomass production for ten microalgal strains

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry depends on the selection of appropriate algal strains. In this study ten species of microalgae were prospected to determine their suitability for oil production: Chaetoceros gracilis, Chaetoceros mulleri, Chlorella vulgaris, Dunaliella sp., Isochrysis sp., Nannochloropsis oculata, Tetraselmis sp., Tetraselmis chui, Tetraselmis tetrathele and Thalassiosira weissflogii. The study was carried out in 3 L glass flasks subjected to constant aeration and controlled artificial illumination and temperature at two different salinities. After harvesting, the extraction of oil was carried out using the Bligh and Dyer method assisted by ultrasound. Results showed that C. gracilis presented the highest oil content and that C. vulgaris presented the highest oil production.     

Specificity of Glycerol for Dark Growth of Prymnesium parvum

SYNOPSIS. Out of 64 nutrients tested, none replaced glycerol for growth in the dark of Prymnesium parvum. Some metabolites enahanced growth in the light, and in the presence of glycerol also in the clark. Good growth with glycerol could be obtained in the absence of CO₂. Survival of cultures in the dark in media without glycerol was prolonged by various nutrients. Thioglycerol in glycerol-containing media inhibited growth in light and darkness. Apparently Prymnesium parvum has a specific glycerol requirement for dark growth.     

Plant biotests of soil and water, polluted with petroleum and petroleum products

Reactiona of higher plants (mustard, oat, rye, salad, dill and barley) and microalgae (Euglena gracilis) on the contamination of soil and water with petroleum and oil products was studied. The germination of seeds was analyzed. The length of sprouts, dry biomass and length of plant roots, as well as the optical density of micro-algal broth culture were determined. Negative effects of soil and water contamination with petroleum and oil products on plant and microalgal parameters examined was shown. After biological destruction of contaminants by an association of destructor strains (Acinetobacter sp., Mycobacterium flavescens and Rhodoccocus sp.), the toxicity of contaminated mediums decreased. The data suggest that the integral toxicity of soil and water contaminated with petroleum and oil products and toxicity change during biodestruction of these pollutants can be analyzed by using plant test organisms.