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Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production., Journal of Extracellular Vesicles, 10 (6), ISSN:2001-3078

Summary The composition of digestive microbiomes is known to be a significant factor in the health of a variety of hosts, including animal livestock. Therefore, it is important to ascertain how readily the microbiome can be significantly altered. To this end, the role of changing diet on the digestive microbiome of the Pacific oyster (Crassostrea gigas) was assessed via weekly faecal sampling. Over the course of 12 weeks, isolated individual oysters were fed either a control diet of Tetraselmis algae (Tet) or a treatment diet which shifted in composition every 4 weeks. Weekly faecal samples from all oysters were taken to characterize their digestive bacterial microbiota. Concurrent weekly sampling of the algal feed cultures was performed to assess the effect of algal microbiomes, independent of the algal type, on the microbiomes observed in the oyster samples. Changing the algal feed was found to be significantly associated with changes in the faecal microbiome over a timescale of weeks between control and treatment groups. No significant differences between individual microbiomes were found within control and treatment groups. This suggests the digestive microbiome of the Pacific oyster can be quickly and reproducibly manipulated.

The Roseobacter-group species Phaeobacter inhibens produces the antibacterial tropodithietic acid (TDA) and the algaecidal roseobacticides with both compound classes sharing part of the same biosynthetic pathway. The purpose of this study was to investigate the production of roseobacticides more broadly in TDA-producing roseobacters and to compare the effect of producers and non-producers on microalgae. Of 33 roseobacters analyzed, roseobacticide production was a unique feature of TDA-producing P. inhibens, P. gallaeciensis and P. piscinae strains. One TDA-producing Phaeobacter, 27-4, did not produce roseobacticides, possibly due to a transposable element. TDA-producing Ruegeria and Pseudovibrio did not produce roseobacticides. Addition of roseobacticide-containing bacterial extracts affected the growth of the microalgae Rhodomonas salina, Thalassiosira pseudonana and Emiliania huxleyi, while growth of Tetraselmis suecica was unaffected. During co-cultivation, growth of E. huxleyi was initially stimulated by the roseobacticide producer DSM 17395, while the subsequent decline in algal cell numbers during senescence was enhanced. Strain 27-4 that does not produce roseobacticides had no effect on algal growth. Both bacterial strains, DSM 17395 and 27-4, grew during co-cultivation presumably utilizing algal exudates. Furthermore, TDA-producing roseobacters have potential as probiotics in marine larviculture and it is promising that the live feed Tetraselmis was unaffected by roseobacticides-containing extracts.

Hydrothermal liquefaction (HTL) is a thermal depolymerization process used to convert wet biomass such as microalgae into bio-crude oil. Four main products, gas, bio-crude oil, an aqueous phase, and solid residue, are generated through HTL. In this study, various HTL conditions were investigated to enhance the phosphorus content in the aqueous phase as well as the yield of bio-crude oil. Tetraselmis sp. was used as the microalgae feedstock, and the product yields according to catalyst type were explored. The phosphate ion (PO43−) content in the aqueous phase was significantly enhanced when acetic acid was added because of the effect of pH. In addition, it was found that both the bio-crude oil yield and the ammonium ion content could be increased by recycling the post-HTL aqueous phase, while the phosphate content was not.

SUMMARY To perform effective measurement of the protein contents, it is necessary to apply the most suitable extraction technique. We compared protein extraction techniques for two microalgal species with entirely different cell wall structures, such as diatom Thalassiosira weissflogii and green flagellate Tetraselmis chuii, using mechanical, physical, and chemical extraction methods. Our results showed that in Th. weissflogii, the control treatment without extraction and mechanical disruption using ultrasonication was sufficient to disrupt its brittle cell wall, resulting in the protein yield of 19.03 and 19.46% of dry weight (DW), respectively. In case of Te. chuii, the chemical extraction using 6% trichloroacetic acid at 95°C was the most suitable extraction method, resulting in the protein yield of 23.78% of DW. Therefore, the most suitable extraction technique must be individually selected for each species depending on their cell wall properties.

Microorganisms present in the rearing water colonize the gut of first feeding larvae and represent the first barrier against opportunistic pathogens. The aim of the experiments presented herein was to standardize a protocol for the management of rearing water and microalgae suitable for the larval rearing of common dentex. In Experiment 1, bacteria–algae interactions were tested using a monospecific microalgal community, Tetraselmis chuii, suitable for nutritional experiments and with known antibacterial activity. In Experiment 2, the evolution of the bacterial community and larval performance (growth and survival) were monitored daily, in three conditions: (1) mature water: T. chuii was added 5 d before the rearing of common dentex larvae, (2) green water: T. chuii was added 1 d before, and (3) clear water: no T. chuii addition. The results show the influence of the presence of T. chuii on the evolution of the bacterial communities, both in terms of bacterial density and morphology, and indicate green water is the most suitable water treatment for management of larval rearing for common dentex. info:eu-repo/semantics/acceptedVersion

Metal risk assessment of industrialized harbors and coastal marine waters requires the application of robust water quality guidelines to determine the likelihood of biological impacts. Currently there is no such guideline available for aluminium in marine waters. A water quality guideline of 24 µg total Al/L has been developed for aluminium in marine waters based on chronic 10% inhibition or effect concentrations (IC10 or EC10) and no-observed-effect concentrations (NOECs) from 11 species (2 literature values and 9 species tested including temperate and tropical species) representing 6 taxonomic groups. The 3 most sensitive species tested were a diatom Ceratoneis closterium (formerly Nitzschia closterium; IC10 = 18 µg Al/L, 72-h growth rate inhibition) < mussel Mytilus edulis plannulatus (EC10 = 250 µg Al/L, 72-h embryo development) < oyster Saccostrea echinata (EC10 = 410 µg Al/L, 48-h embryo development). Toxicity to these species was the result of the dissolved aluminium forms of aluminate (Al(OH4 (-) ) and aluminium hydroxide (Al(OH)3 (0) ) although both dissolved, and particulate aluminium contributed to toxicity in the diatom Minutocellus polymorphus and green alga Dunaliella tertiolecta. In contrast, aluminium toxicity to the green flagellate alga Tetraselmis sp. was the result of particulate aluminium only. Four species, a brown macroalga (Hormosira banksii), sea urchin embryo (Heliocidaris tuberculata), and 2 juvenile fish species (Lates calcarifer and Acanthochromis polyacanthus), were not adversely affected at the highest test concentration used.

The effect of light quality on cell size and cell cycle, growth rate, productivity, photosynthetic efficiency and biomass composition of the marine prasinophyte Tetraselmis suecica F&M-M33 grown in 2-L flat panel photobioreactors illuminated with light emitting diodes (LEDs) of different colors was investigated. Biomass productivity and photosynthetic efficiency were comparable between white and red light, while under blue and green light productivity decreased to less than half and photosynthetic efficiency to about one third. Differences in cell size and number correlated with the cell cycle phase. Under red light cells were smaller and more motile. Chlorophyll content was strongly reduced with red and enhanced with blue light, while carotenoids and gross biomass composition were not affected by light quality. The eicosapentaenoic acid content increased under red light. Red light can substitute white light without affecting productivity of T. suecica F&M-M33, leading to smaller and more motile cells and increased eicosapentaenoic acid content. Red LEDs can thus be profitably used for the production of this microalga for aquaculture.

The effects of two microencapsulated feeds were evaluated on development, growth, survival, proteolytic activity, and biochemical composition of white shrimp, Litopenaeus vannamei, larvae. The treatments were: (1) basal microcapsules (BM), (2) microcapsules containing krill hydrolysate (BMK), and (3) live food control (LFC: Artemia franciscana nauplii) with all treatments receiving algae (Chaetoceros ceratosporum and Tetraselmis chuii). No significant differences were found in development index and survival among larvae. Growth rate was significantly higher in larvae fed LFC (15 +/- 0.06%/d) as compared with those offered the BM diet (7.5 +/- 0.5%/d) with the BMK (11 +/- 0.04%/d) treatment producing intermediate results. The activity of total proteases and chymotrypsin decreased significantly after Mysis I (MI) in larvae fed LFC or BMK. Protein content of larvae increased significantly toward PL1. The acylglycerides content in MIII fed on LFC (2.3 +/- 0.2%/dw) was higher than that MI fed BM (1 +/- 0.01%/dw). No difference was observed in the cholesterol (CH) content of the larvae. The acylglycerides/protein and cholesterol/protein ratios showed a decreasing pattern between MI and PL1, indicating that these two ratios were related to ontogenetic shifts. These results demonstrate improvements in microparticulate diets when krill hydrolysates are included in the formulation.

Classical models of prey-predator interactions assume that per capita prey consumption is dependent on prey density alone and that prey consumption (functional response) and consumer proliferation (numerical response) operate on the same timescales and without time lags. Several modifications have been proposed for resolving this timescale discrepancy, including variants where the functional response depends on both prey and predator densities. A microcosm system with the rotifer Brachionus 'Nevada' feeding on the prasinophyte Tetraselmis sp. showed significant (P0.0005) increases in steady-state biomasses of both prey and predators with increasing carrying capacity (represented by total phosphorus of the growth medium), which is inconsistent with predictions based on the traditional prey-only-dependent functional response. We provide data indicating that surfaces where the predator can attach provide a high-quality habitat for rotifers, which can result in a predator-dependent functional response. We also show that partitioning between the attached and free-swimming habitats was fast compared to the timescale of the numerical response. When attached to surfaces, rotifers maximized net energy gain by avoiding the high cost of swimming and by increased food capture due to reduced viscous drag. A mathematical model with prey-dependent functional response and wall-attached and free-swimming fractions of the population describes our data adequately. We discuss the implications of this finding for extrapolating microcosm experiments to systems with other surface-to-volume ratios, and to what extent our findings may apply to other popular model organisms for prey-predator interaction.

Four different seaweed extracts were employed as the dyes of dye sensitized solar cells (DSSCs) to investigate the light-electron efficiency. The sensitizers, extracted from Nannochloropsis spp., Tetraselmis spp., Gracilaria spp., and Ulvales spp., showed their light-electronic transfer ability in different light intensities. Among them, Ulvales output a higher light-voltage, about 0.4 V. The output voltage increased when light intensity increased. Gracilaria extract produced a higher output voltage at 35 Lux, but its output voltage decreased over 500 Lux. The sensitizers extracted from these seaweeds had monochromatic incident photon-to-electron conversion efficiencies (IPCE) between 23-61% in 220-260 nm wavelengths. Among them, Ulvales output higher IPCE than Tetraselmis and Nannochloropsis. SEM analysis of DSSC surfaces revealed that the efficiency of seaweed DSSCs was governed by chlorophyll size. The chlorophyll particle size of Ulvales spp. was the largest. The chlorophyll particle size of Gracilaria spp. was the smallest and yielded the lowest IPCE.

Tetraselmis sp. and Nannochloropsis oculata, cultivated in industrial-scale bioreactors, produced 2.33 and 2.44% w/w lipid (calculated as the sum of fatty acid methyl esters) in dry biomass, respectively. These lipids contained higher amounts of neutral lipids and glycolipids plus sphingolipids, than phospholipids. Lipids of Tetraselmis sp. were characterized by the presence of eicosapentaenoic acid (that was located mainly in phospholipids), and octadecatetraenoic acid (that was equally distributed among lipid fractions), while these fatty acids were completely absent in N. oculata lipids. Additionally, lipids produced by 16 newly isolated strains from Greek aquatic environments (cultivated in flask reactors) were studied. The highest percentage of lipids was found in Prorocentrum triestinum (3.69% w/w) while the lowest in Prymnesium parvum (0.47% w/w). Several strains produced lipids rich in eicosapentaenoic and docosahexaenoic acids. For instance, docosahexaenoic acid was found in high percentages in lipids of Amphidinium sp. S1, P. parvum, Prorocentrum minimum and P. triestinum, while lipids produced by Asterionella sp. (?) S2 contained eicosapentaenoic acid in high concentration. These lipids, containing ω-3-long-chain polyunsaturated fatty acids, have important applications in the food and pharmaceutical industries and in aquaculture.

Red shrimp, Farfantepenaeus brasiliensis, larvae's response to different concentrations of live foods (diatoms Chaetoceros gracilis: 20-100 x 103 cells/mL; flagellate Tetraselmis chuii: 2-10 x 103 cells/mL and Artemia nauplii [NA]: 1-5 NA/mL) was investigated in three experiments. Experimental assessments were based on four variables: survival rate, weight gain, development index (DI), and resistance to salinity stress. A combination of C. gracilis 80 x 103 cells/mL, T. chuii 2 x 103 cells/mL, and Artemia 4 NA/mL provided the best experimental response. Specifically, F. brasiliensis larvae fed with the above-mentioned optimal concentrations of microalgae and Artemia grew faster and reached the postlarval stage in less time (168 h) than larvae in other feeding regimes evaluated. The effect of C. gracilis concentration on larval growth, survival, and the DI demonstrated that microalgae-based foods could be a highly productive alternative to more traditional aquaculture feeding regimes.

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale-up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1-, f/2-, and K-medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9-4.8 g m(-2) day(-1) in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1-medium. The dry biomass productivity of Tetraselmis sp. (33.1-45.0 g m(-2) day(-1)) exceeded that of the other species by a factor 2-10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large-scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems.

9 páginas, 5 tablas, 2 figuras, The pigment composition of six species of Tetraselmis (Prasinophyceae) was analyzed using improved HPLC methods. All pigment extracts showed three peaks corresponding to unknown carotenoids. The isolated pigments were analyzed using UV–Vis spectroscopy, electrospray ionization–mass spectrometry (ESI–MS), and when carotenoid esters were suspected, gas chromatography–mass spectrometry (GC–MS) of the methyl ester and dimethyloxazoline derivative of the corresponding fatty acid. The new pigments were determined to be loroxanthin, loroxanthin 19-(2-decenoate), and loroxanthin 19-(2-dodecenoate); this is the first time these pigments have been described in the genus Tetraselmis. Moreover, this is the first report of esterification of 2-decenoic acid to loroxanthin. The relative contents of these pigments depended on the light regime, with the lowest proportions measured at the highest photon flux density assayed. The implications of the identification of these pigments in the genus Tetraselmis for the pigment types previously described in the class Prasinophyceae are discussed, This work was supported by Xunta de Galicia through project PGIDT99MAR50102.

Experiments conducted with Penaeus aztecus, P. setiferus and P. vannamei compared survival, metamorphosis and dry weight growth of larvae fed a variety of algal and animal food regimes. The major objectives were: (a) comparison of the nematode Panagrellus redivivus and the brine shrimp Artemia as animal food sources and (b) comparison of effects of feeding diatoms (Skeletonema costatum and Chaetoceros gracilis) vs phytoflagellates (Isochrysis sp. and Tetraselmis chuii) with nematodes or brine shrimp nauplii added to the diet starting at the protozoea-two or mysis-one substage. Specific feeding regimes were identified for each shrimp species tested which would allow for total or partial replacement of Artemia as the animal food source, without reducing production results. However, Panagrellus redivivus was found to be less versatile than Artemia nauplii in terms of overall nutritional value to the shrimp larvae. The superiority of using two diatom species vs two phytoflagellate species as an algal food source for penaeid shrimp larvae demonstrated in earlier experiments was confirmed. The results strongly suggest that evaluation of rearing regimes used for production of penaeid postlarvae should include growth in terms of dry weight, as well as survival and rate of metamorphosis.

Tetraselmis sp., a green flagellate, shows promise as a good source for larval crustaceans. This paper discusses culture techniques. Typical densities produced in the mass-culture tanks ranged from 275,000 to 450,000 cells per ml.

Studies on spawning and larval culture in the purple-hinge rock scallop (Hinnites multirugosus) are described together with information on growth of juveniles and adults. Gravid individuals were collected from field populations throughout most of the year. These produced viable eggs when induced to spawn in the laboratory. Larval development appeared normal over the range 8–18°C (optimal for culture, 14–18°C). Acceptable growth and survival were achieved on a mixed diet of Isochrysis, Monochrysis, Tetraselmis and Skeletonema. Metamorphosis began in larvae 200 μm in shell diameter (age, 3 weeks). Most rapidly growing post-larvae reached 1.8 mm at two months (15–18°C). Pre-cementing juveniles held in cages in Mission Bay, San Diego, averaged approximately 8 mm/mo during summer (18–22°C) and half that rate throughout the rest of the year (13–18°C). Individuals 1.5 to 3.0 cm in October, 1975, reached shell diameters of 5.0 to 7.5 cm one year later.

Twenty-five spawning attempts on the most reproductively developed fish were conducted on conditioned and unconditioned Florida pompano (Trachinotus carolinus). Human chorionic gonadotropin (HCG) was used to stimulate ovarian development. Dosage levels of .55 IU/g and .275 IU/g were usually administered 24 to 48 hours apart. Strip spawning and semi-natural spawning were used, Spawning occurred 7 to 208 hours after initial injection depending on initial oocyte size. Optimum oocyte size (580–718 μ) produced spawning in 30–40 hours. A close correlation exists between rate of fertilization and oocyte growth during the first 16–25 hours. Fertilized eggs may shrink as much as 16% within one hour after fertilization. Two successive batches of pompano were reared to juvenile stage in 23 days. Larvae were fed a mixed diet of natural plankton (66 μ mesh net), protozoans, rotifers (Brachionus plicatilus) and copepod nauplii (Oithona sp.). Water was initially conditioned with 40 liters of dense algae (Dunaliella sp. and Tetraselmis sp.). Water quality was controlled by partial exchanges from a 40,000-liter biologically filtered system. Eighteen-hour lighting was used. Larvae developed edema at 8–10 days and, combined with low aeration, reduced survival to only 300 juveniles.

Various methodological approaches are being used to characterize Symbiodinium spp isolated from Marginopora vertebralis and Amphisorus hemprichii and Amphisorus sp. from the Indo-pacific. While most of the cells in culture are non-motile vegetative cells, dinospores occur in batch cultures. We find consistant variance among the isolates in the duration and time of appearance of motile forms. Nitrate (0.146 mM) supports higher populations of the isolates than NH4 (0.146 mM). Higher concentrations of NH4 inhibit growth. Although Fensome et al. (1993) characterized the Order Suessiales (and Family Symbiodiniaceae) as gymnnodiniphycideans in which the amphiesmal vesicals are arranged in 7–10 longitudinal series we were unable to detect any plates on the surfaces of our isolates in the SEM. We were unsuccessful in revealing plates on non-motile forms by freeze fracture but they were present after treatment with various chemical agents (e.g. H2O2, HOCl−) and enzymes (cellulase, chitinase). They are not polygonal or arranged in the patterns anticipated for the Symbiodiniaceae. Polyclonal antibody studies of the Symbiodinium-like isolates are in agreement with molecular systematic studies in the sense that there is a common relation. The foram endosymbiotic dinoflagellates share some common surface antigens with each other and with the few invertebrate isolates we have tested. Studies of the foraminiferal symbionts' plastids revealed considerable morphological diversity. We isolated a diversity of endosymbionts from the mantle of Tridacna maxima from the Red Sea and the Marshall Islands. These included Symbiodinium, Amphidinium, Tetraselmis, and an unidentified chlorophyte.

The prasinophyte genera Scherffelia and Tetraselmis are the only genera that form a cell wall by an extracellular fusion of scales called a theca. We established a protocol for the production of protoplasts from Scherffelia dubia Pascher emend. Melkonian et Preisig using 3 mM Ellman's reagent (5,5′-dithio-bis-2-nitrobenozoic acid [DTNB]). Protoplasts analyzed by EM lacked flagella and thecae but were otherwise similar to control cells. In response to treatment with DTNB, many protoplasts synthesized new thecal scales in the Golgi apparatus, indicating that cells attempted to regenerate new cell walls. However, complete regeneration of the thecae only occurred once DTNB was washed out from the medium. At higher DTNB concentrations (5 mM), two protoplasts were found within the parental cell wall and scales accumulated between the plasma membrane of the protoplasts and the original theca but failed to form a new theca.

Negative effects of prey species on consumers could be due to deterrence, nutritional insufficiency, or toxicity of the prey. These effects can be discerned in experiments in which the suspect prey is offered to the consumers in a food mixture containing another prey item that is not toxic and in which the ingestion rates on the prey and the grazers’ responses (e.g., egg production) are measured. We used this framework to determine whether several algae that have been reported to have harmful effects on grazers (Prorocentrum minimum, low- and high-toxin Alexandrium sp., Heterosigma carterae, Thalassiosira rotula, and Phaeodactylum tricornutum ) are toxic to females of the calanoid copepod Acartia tonsa. Ingestion, egg production, and egg-hatching rates were measured for A. tonsa offered sole diets of the suspect alga and mixed diets containing the suspect alga and a control alga (the green flagellate Tetraselmis sp.) at an ecologically relevant concentration (250 m gCL 21 ) and duration (3 d). With the exception of the Alexandrium strain with the high-toxin content (16.3 pgSaxitoxin [pgSTX] equivalents cell 21 ), none of the diets studied can be considered toxic. The high-toxin Alexandriumreduced A. tonsa’s total ingestion rate, and thus egg production, as the proportion of Alexandrium increased in the diet. A. tonsa exhibited significantly reduced ingestion and egg production rates when feeding on sole food diets of H. carterae and P. tricornutum relative to the mixed food diets. However, a comparison of ingestion rates among the diet mixtures revealed that H. carterae and P. tricornutum acted as feeding deterrents when provided as sole foods. These results stress the importance of using mixed food diets when examining putative toxic effects of preys on consumers. Reports of putatively harmful effects of phytoplankton on grazers ranging from feeding inhibition and physiological incapacitation to reductions in egg production and hatching rates are common (reviewed in Turner and Tester 1997). The broader ecological significance of these effects depends on whether they are severe enough to reduce secondary production or the grazers’ impact on algal population growth. Many studies dealing with harmful effects of phytoplankton on grazers have focused only on feeding activity, which has been found to be reduced by some phytoplankters (Ives 1987; Uye and Takamatsu 1990; Teegarden 1999). Other studies have focused on the effects on egg production and hatching rates without measuring ingestion rates (Ianora and Poulet 1993; Laabir et al. 1995; Uye 1996; Ban et al. 1997). These limitations hinder our ability to evaluate fully and predict the ecological effects of putatively harmful algae in natural systems. The harmful nature of phytoplankters is typically assumed to be due to their toxicity. Determining phytoplankton toxicity is straightforward when the effects are lethal, which is typically not the case. Moreover, a negative effect of a phytoplankter on a grazer relative to some other diet—such as

Se is present in multiple oxidation states in nature, each of which has unique chemical and biological reactivities. As a consequence, the rate of Se incorporation into food webs or its role as either a limiting nutrient or a toxic substance is a function of complex biogeochemistry. In particular, little is understood about the accumulation of dissolved organic selenides by phyto- or bacterioplankton. We assessed the bioavailability of dissolved organic selenides to marine and estuarine phytoplankton by presenting various algal species with filtered lysates of the diatom, Thalassiosira pseudonana, grown on media amended with radiolabeled selenite (75Se[IV]). Species known to accumulate selenite effectively also accumulated Se from radiolabeled cell lysates and to approximately the same extent. When exposed to a 4.5-nM solution of lysate Se, T. pseudonana, Heterocapsa triquetra (Dinophyceae), Tetraselmis levis (Prasinophyceae), Synechococcus bacillus (Cyanobacteria), and Dunaliella tertiolecta (Chlorophyceae) incorporated 42%-53%, 42%, 30%, 32%, and 4% of the dissolved Se, respectively. Se cell contents of T. pseudonana, T. levis, and D. tertiolecta grown in media containing 4.5 nM lysate Se were very similar to Se content of cells grown in 4.5 nM selenite. Our results suggest that recycling of Se(-II) may be more important than previously thought. Consequently, uptake of organic selenides by phytoplankton may need to be considered in models predicting Se incorporation into aquatic food webs, given that it is a significant fraction of dissolved Se in estuarine and oceanic waters.

To evaluate the use of pigments as tracers for determining copepod grazing rates and selectivity, we examined the stability of several biomarker pigments during copepod feeding incubations. During these incubations, we measured changes in phytoplankton-derived chlorophylls and carotenoids in the particulate and dissolved pools. Budgets were calculated to determine changes in pigment concentrations in the food, copepod fecal pellets, copepod guts, and dissolved/colloidal fraction. For each of six algal diets, adult female Acartia tonsa copepods were fed limiting and saturating food concentrations, approximately 100 and 500 µg C L−1 , respectively. Thalassiosira weissflogii, Rhodomonas lens, Chroomonas salina, Dunaliella tertiolecta, and two Tetraselmis strains were used in the feeding experiments to investigate the fate of fucoxanthin, alloxanthin, lutein, chlorophyll (Chl) b, and Chl a. In all experiments using saturating food concentrations, the dissolved/colloidal pool contained no more than 3% (usually less than 1%) of any pigment, whereas in experiments using limiting food conditions, pigments were undetectable in the dissolved/colloidal pool. Pheopigments were present in fecal pellets and copepod guts in most of the experiments. Chl a destruction (conversion to colorless products) was variable among the different experiments, depending on algal species and food concentration and, in most cases, Chl a was destroyed to a greater extent than the carotenoids. In all cases, pigment destruction was higher when copepods were fed limiting rather than saturating food concentrations. These data attribute the variability in pigment destruction to algal species and concentration, and suggest caution when pigments are used as tracers of herbivory. In such studies, assumptions about conservative behavior, even for the carotenoids, would need to be verified for each set of experimental conditions and grazers.

SUMMARY The Culture Collection of Algae and Protozoa (CCAP) and the National Institute for Environmental Studies (NIES) act as service collections in the UK and Japan, respectively. Since 1991, the collections have collaborated on a wide range of topics of mutual interest. These include: algal taxonomy, bio-informatics, collection management, conservation of microbial biodiversity, cryopreservation, and cyanobacterial toxin research. The main activities have focused on the re-examination of cyanobacterial isolates housed in both collections and cryopreservation research. The preliminary data generated in the survey of cyanobacterial strains were used in the production of the latest CCAP catalogue and database. This project is ongoing at NIES and forms a key component of a major project on cyanobacterial taxonomy. To date, many protists are apparently freeze-recalci-trant. A primary mode of lethal cell injury is the formation of intracellular ice. In this study, intracellular ice formation was observed in Tetraselmis suecica (Ky-lin) Butcher under suboptimal cooling conditions, Tetraselmis chui Butcher under both fast and slow cooling and in both pigmented and achlorophylous strains of Euglena gracilis Klebs. These findings have significant implications for the development of successful cryopreservation strategies. In addition to the above activities, a variety of algal strains have been examined in taxonomic and ultra-structure research projects and cyanobacterial cultures have been screened for novel toxins.

The drinking rate of turbot larvae increased from 14 to 120 nl larva-1 h-1 from day 2 to 11 after hatching, which gave a slightly increased specific drinking rate (calculated per biomass) from day 2 to 7 (0·8–1·9 nl μg carbon-1 h-1. The clearance rate of both algae and bacteria was 10–100 times higher than the drinking rate, which indicated that the larvae had an active uptake of both algae and bacteria. On day 2 and 4 after hatching highest clearance rate was observed for Tetraselmis sp. On day 6 about the same clearance rate was observed for bacteria, Isochrysis galbana and Tetraselmis sp. Until day 4 the turbot larvae had a higher ingestion rate of Tetraselmis sp. than of I. galbana, whereas on day 6 the rates were similar (28–41 ng carbon larvae-1 h-1). The assimilation efficiency was somewhat higher for I. galbana than for Tetraselmis sp., and on day 6 the assimilated algae constituted 1·5 and 0·9% of the larval biomass for I. galbana and Tetraselmis sp., respectively.

— The impact of ultraviolet-B radiation (UVBR) on the biosynthesis and the composition of intracellular dissolved free and combined amino acids was examined in nitrate-replete and nitrate-deficient cultures of Tetraselmis sp. Several similarities were observed in the response of Tetraselmis sp. to nitrogen deficiency and exposure to UVBR, in support of a view that UVBR affects amino acid synthesis in phytoplankton via its inhibitory effects on nitrogen assimilation into the cells. At levels that still permitted the uptake of carbon into the cells, both nitrogen deficiency and UVBR exposure resulted in a reduction in the overall rates of carbon incorporated into amino acids, an increase in the absolute concentrations of amino acids within the intracellular dissolved free amino acid (INDFAA) pool and a decrease in the total cellular amino acid (TCAA) pool. An examination of the patterns of carbon assimilation into individual amino acids in cells exposed to UVBR revealed similarities with the patterns in cells subject to nitrogen deficiency. The most conspicuous changes from the controls included an increased incorporation of 13C into glutamic' acid (glutamic acid + glutamine) and aspartic acid and a marked reduction into alanine and valine. Changes in the concentrations of amino acids within the INDFAA and TCAA pools were also similar in nitrate-deficient and UVBR-exposed cells and resembled the carbon assimilation patterns. These results strongly suggest that UVBR-induced changes in the biosynthesis and composition of amino acids are probably via its suppression of nitrogen assimilation into the cells.

Phaeodactylum tricornutum and Chaetoceros sp. (Badllariophyceae), Isochrysis galbana (clone T-Iso) and Pavlova lutheri (Prymnesiophyceae), Nannochloris atomus (Chlorophyceae), Tetraselmis sp. (Prasinophyceae), and Gymnodinum sp. (Dinophyceae) were cultured at different extents of nutrient-limited growth: 50 and 5% of μmax. The lipid content of the algae was in the range 8.3–29.5% of dry matter and was generally higher in the Prymnesiophyceae than in the Prasinophyceae and the Chlorophyceae. Increasing extent of phosphorus limitation resulted in increased lipid content in the Bacillariophyceae and Prymnesiophyceae and decreased lipid content in the green flagellates N. atomus and Tetraselmis sp. The fatty acid composition of the algae showed taxonomic conformity, especially for the Bacillariophyceae, where the major fatty adds were 14:0, 16:0, 16:1, and 20:5n-3. These fatty acids were dominant also in the Prymnesiophyceae together with 22:6n-3. An exception was I. galbana, in which 18:1 was the major monounsaturated fatty add and 20:5n-3 was absent. The fatty acids of N. atomus and Tetraselmis sp. varied somewhat, but 16:0, 16:1, 18:1, 18:3n-3, and 20:5n-3 were most abundant. Gymnodinum sp. contained mainly 16:0, 18:4n-3, 20: 5n-3, and 22:6n-3. An increased level of nutrient limitation (probably phosphorus) resulted in a higher relative content of 16:0 and 18:1 and a lower relative content of 18:4n-3, 20:5n-3, and 22:6n-3. The nutrient limitation probably reduced the synthesis of n-3 polyunsaturated fatty acids.

Knowledge of tolerance to cryoprotectants is important in determining viability after biological freezing of algae. Six taxonomically divers marine microalgae were evaluated for their tolerance to the widely used cryoprotectants dimethyl sulfoxide (DMSO) and methanol. Tetraselmis chuii Butcher survived exposure to 30% (v/v) DMSO and 25% methanol for periods of up to 4 h. All other species were more sensitive to high concentrations of these cryoprotectants. DMSO was lethal at 25% after a 15-min exposure of Rhodomonas baltica Karsten, Isochrysis aff. galbana (strain T-ISO) Parke, and Nannochloropsis gaditana Lubian

The drinking rate of water, and the ingestion- and assimilation rates of algae in yolk-sac larvae of Atlantic halibut were examined by use of3H-dextran and14C-labelled algae. The drinking rate throughout the yolk-sac stage was in the range of 7–160 nl per larva h−1, with a slight increase towards the end of the period. The ingestion rate of algae (Tetraselmis sp.) was very low before day 30 and showed a peak between day 43 and 48 at 5° C. The assimilation efficiency of the algae was in the range of 1-5%. The clearance rate of algae was 100–1000 times higher than the drinking rate, and was correlated to the distance between the gill arches. Bigger cells were more effectively retained than smaller ones. The results suggested that the larvae acted as filter feeders from day 30 up to the point when they are expected to start feeding on bigger prey.

Centrin is a major protein of the contactile striated flagellar roots of the green alga Tetraselmis striata. We present a newly modified procedure for the preparation of centrin in sufficient quantity and purity to allow for detailed biochemical characterization. We establish that centrin purified by differential solubility, followed by phenyl-Sepharose and DEAE-Sephacel chromatography is identical with the protein extracted directly from striated flagellar roots with regard to molecular weight, isoelectric point, and calcium-dependent behavior in SDS-PAGE. We also compare the biochemical properties of purified centrin with calmodulin isolated from Tetraselmis and calmodulin isolated from mammalian brain. Centrin can be fully distinguished from either algal or mammalian calmodulin on the basis of molecular weight, isoelectric point, calcium-dependent behavior in SDS-PAGE, proteolytic peptide maps, amino acid composition, ability to activate bovine brain phosphodiesterase, and reactivity with specific antibodies.

The phylogenetic distribution of photosystem I-associated polypeptides was assessed by immunoblotting algal thylakoid membrane polypeptides with antisera generated against the P700-chlorophyll a protein (CC I) and a photosystem I light-harvesting chlorophyll-protein (LHC Ib). Polypeptides cross-reacting with the CC I apoprotein were found in 20 species representing four classes of unicellular algae. Polypeptides sharing antigenicity with spinach LHC Ib were observed only in algal species containing chlorophyll b. Tetraselmis spp. (Pleurastrophyceae), rich in chlorophyll b (Chl a:b 1.2), exhibited marked heterogeneity in the composition of their CC I and LHC Ib cross-reactive polypeptides. When immunoblotted with antisera against CC I, all Tetraselmis clones examined exhibited a 25-kD polypeptide in greater abundance than the 58-kD CC I apoprotein characteristic of higher plants and other green algal thylakoids. Three Tetraselmis clones (RG 6, RG 11, and RG 12) exhibited an 81-kD polypeptide with strong antigenicity toward the LHC Ib antisera, in contrast to the 17- to 24-kD cross-reactive polypeptides found in spinach, green algae, and one Tetraselmis clone (RG 5). Associated with the unique photosystem I polypeptide composition in Tetraselmis spp., Chl: P700 ratios for the group are 2–5 times greater than those observed for higher plants or other green algae. The chlorophyll b enrichment, unusual composition of photosystem I cross-reactive polypeptides, and heterogeneity of these polypeptides within isolates of Tetraselmis might make this genus useful for investigations of the functional organization of chlorophyll b in light-harvesting systems. These features also support the view of an alternative phyletic origin for the Pleurastrophyceae.

Fluorescent labeling of the flagellar apparatus of Tetraselmis (Prasinophyceae) and Dunaliella (Polyblepharidaceae, Chlorophyceae) were successfully performed using fluorescein isothiocyanate–labeled lectins from Arachis hypogaea and Glycine maxima. These lectins specifically bound to the flagella and kinetosome of the cell but did not bind to the cell surface. Lectin binding on the flagellar apparatus remained constant under different culture media, temperatures, irradiances, cell division cycle, and culture aging. All the Tetraselmis and Dunaliella analyzed (five species, 20 clones) showed intense labeling of the flagellar apparatus. In contrast, no other species analyzed (46 clones of 25 species from four classes) showed binding to their flagellar apparatus. After the lectin treatment, many cells remained alive, and they were able to swim with the flagellar apparatus intensely labeled. The lectin binding indicates that the flagella and kinetosome of Tetraselmis are rich in Gal and GalNH2 moieties and that the flagella of Dunaliella are rich in Gal and GalNAc moieties. Apparently, this feature seems to be specific to these species.

In clonal culture Scherffelia dubia (Perty) Pascher shows a considerable range of morphological variation, with different cell shapes being produced including those typical for three other species of Scherffelia Pascher (S. opisthostigma Skuja, S. ovata Pascher, S. pelagica Skuja) and one species of Tetraselmis Stein (T. bichlora Ettl & Ettl). It is concluded these taxa should be reduced to the synonymy of S. dubia, the type species of Scherffelia. The general cell morphology of S. dubia is described in detail, including a serial section and absolute configuration analysis of the flagellar apparatus. S. dubia is compared with species of Tetraselmis of which ultrastructural details are known and it is shown that both genera are very similar but the generic distinction is nevertheless justified. Main features of distinction include the pyrenoid (present in Tetraselmis but lacking in Scherffelia) and the chloroplast (single and more or less cup-shaped in Tetraselmis, two separate lateral chloroplasts in Scherffelia). Both genera are accommodated in the same family, the correct name being Chlorodendraceae and not Tetraselmidaceae or Platymonadaceae which have often been used.

SUMMARY The relationship between assimilation of nitrogen and animating activities of glutamine synthetase (GS), NADH-glutamate dehydrogenase (GDH) and NADPH-GDH in ammonium-grown Chlamydomonas pulsatilla Wollen-weber and ammonium- and nitrate-grown Brachiomonas submarina Bohlin and Tetraselmis succica (Kylin) Butcher was investigated. The three flagellates show minor changes (15–60%) in enzyme activities when ammonium is replaced by nitrate as the nitrogen source or the concentration of nitrogen is lowered from 2 to 0·2 mM. The activities of NADH-GDH and NADPH-GDH in the three flagellates do not appear to be high enough to make a significant contribution to ammonium assimilation in the presence of a highly active GS. Fast protein liquid chromatography (FPLC) of GS revealed the presence of two distinct molecular forms, designated as GS1 and GS2, in C. pulsatilla and T. suecica, whereas only a single GS peak designated as GS2 was detected in B. submarina. Both GS1 and GS2 are stabilized by sorbitol and glycinebetaine in media employed for enzyme extraction and FPLC fractionation. The presence of thiol reagents is essential for GS2 stability, but inhibits GS1 activity. GS2 is by far the more active of the two isoenzymes in C. pulsatilla and T. suecica. This isoenzyme, therefore, appears to be the major port of entry of ammonium in all three flagellates.

Characteristics of the growth of PhaeodactyZum in culture in fertilized sea water in 1,000-L concrete tanks are dcscrihed. Successful experiments showed a constant pattern of events as follows: 1) active growth of PhaeocZnctyZum, 2) reduction in growth rate of Phaeodnctylum, 3 ) period of constant cell density, or rapid decline, 4 ) rcgcneration period, and, 5) second period of active growth, Growth of Phaeodactylum shows seasonal variation, but final population density reached is more or less constant. N and P utilization follow parallel courses, the mean ratio of N:P uptake during the 1st growth period bdng 6.5 2 0.44: I. During growth, inorganic P is incorporated in the cells as particulate organic P. Following dcclinc of PhaeocZactyZum dissolved organic P incrcascs, and later, inorganic P is regencratcd and may be reutilized during the 2nd growth phase. Decline of Phneodactylum is normally associated with increase of the heterotroph Mows. Thcrc was a close relationship bctwecn peak numbers of Mows and the number of Phaeotlactylum in the previous peak. The second growth phase consisted either of one or more chlorophycean flagcllatcs, or of Phaeoductylum. Estimates of production based on 02 bottle exposures, change in inorganic P, change in pH, and change in packed ccl1 volume agree closely, although initial uptake of P may bc excessive. Estimates from change in inorganic P indicate a seasonal variation in the rate of organic production of up to 2.2 gC/m”/24 hr in March, 4.9 gC/m2/24 hr in May-August, and 2.0 gC/m”/24 hr in October, November. For Tetruselmis production rates up to 5.2 gC/m2/24 hr were recorded. The efficiency of utilization of solar energy for Phaeoductykm culture ranged from 3.0 to 4.4% with a mean for 5 cstimatcs of 3.7%. For Tetraselmis values of 1.0-1.3s wcrc obtained.

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.

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.