Variations in pigment contents of the diatom Phaeodactylum tricornutum during growth

Changes in the amounts of chlorophyll a, chlorophyll c, fucoxanthin, diadinoxanthin and -carotene were determined during Phaeodactylum tricornutum growth. The transition of the culture from the logarithmic to the stationary phase is accompanied by an increase in the carotenoids: chlorophyll a ratio, associated with variations in the percentage of individual carotenoids. While fucoxanthin content decreases with the age of the culture, diadinoxanthin content increases and -carotene remains almost constant. The furanoid isomer of diadinoxanthin, absent during logarithmic growth, appears increasingly during the nutrient-deficient period. Changes in the amounts of chlorophyll a, chlorophyll c and fucoxanthin are quite similar.     

Assessing the toxicity of chemical compounds associated with marine land-based fish farms: The use of mini-scale microalgal toxicity tests

Many chemicals that are currently used in aquaculture have not been evaluated with regard to their specific effects on the aquatic environment. In the present study, the toxic effects of several chemicals associated with land-based marine fish farming activities were assessed using two species of marine microalgae (Phaeodactylum tricornutum and Isochrysis galbana). Mini-scale toxicity tests were performed with six antibiotics (amoxicillin, ampicillin, flumequine, oxytetracycline, streptomycin and sulfadiazine) and two disinfectants (formaldehyde and hypochlorite). Amoxicillin and streptomycin did not exert toxic effects. Sulfadiazine was the most toxic chemical; the EC₅₀ values were 0.11 mg/L and 1.44 mg/L for P. tricornutum and I. galbana respectively. As expected, the disinfectants displayed high toxicity, and P. tricornutum was particularly sensitive to these compounds. Although the differences in microalgal sensitivity depended on the chemical considered, both species were highly sensitive to most of the compounds tested. We recommend the inclusion of mini-scale microalgal toxicity tests in environmental risk assessment (ERA) and environmental monitoring plans because they are cost-effective and rapid.     

Growth and competition of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. I. Nutrient effects

Two marine diatoms, Phaeodactylum tricornutum (Bohlin) and Thalassiosira pseudonana (Hasle and Heimdal), were grown both separately and together in batch cultures on a mixture of waste water and seawater enriched with different components of f medium. At 17°C, the maximum division rates of the two species were statistically indistinguishable. The waste water-seawater mixture used proved to have insufficient Si, relative to N and P, for the growth of T. pseudonana, which requires approximately 5x10^-14 g-at Si cell^-1 to divide at a maximum rate. P. tricornutum, on the other hand, although capable of taking up nearly 9x10^-15 g-at Si cell^-1, could sustain maximum rates of division with 4.3x10^-18 g-at Si cell^-1 or less. No allelopathic interaction between the two species could be detected. We conclude that P. tricornutum enjoys a considerable competitive advantage over T. pseudonana in a waste water-seawater-based mariculture system that is not supplemented with Si. Although Si proved necessary for T. pseudonana to complete more successfully with the other diatom, the presence of excess amounts of Si is not necessarily sufficient for the maintenance of T. pseudonana in mixed continuous culture with P. tricornutum: other factors, such as light-related or photoperiod-related growth response, are believed to determine the ultimate outcome of competition between these algae in light-limited continuous culture.Contribution No. 3999, from the Woods Hole Oceanographic Institution.Communicated by M.R. Tripp, Newark     

Effects of ethyl 2-methyl acetoacetate (EMA) on the growth of Phaeodactylum tricornutum and Skeletonema costatum

We investigated the effects of ethyl 2-methyl acetoacetate (EMA) on growth of the marine diatom algae Phaeodactylum tricornutum (P. tricornutum) and Skeletonema costatum (S. costatum). Growth of P. tricornutum was significantly inhibited by the minimum concentration (3.5 mmol·L ^?1) of EMA at lower initial algal densities (IADs) (3.6×10⁴ and 3.3×10⁵ cells·mL ^?1). However, at the highest IAD, significant growth inhibition was found at above 7 mmol·L ^?1 of EMA exposure. In S. costatum, EMA concentrations of 10.5 mmol·L ^?1 or more significantly inhibited growth at lower IAD (3×10⁴ and 1.8×10⁵ cells·mL ^?1); at the highest IAD, only EMA concentrations above 14 mmol·L ^?1 obviously inhibited the growth of S. costatum. Changes in specific growth rates and pigment were consistent with algal growth, but only at higher EMA concentrations or lower IAD values was the ratio of chlorophyll a (Chla) to carotenoid significantly lower than the control. Medium effective concentration (EC ₅₀) values were in the order 4.07, 8.03 and 12.27 mmol·L ^?1 for P. tricornutum and 7.48, 11.92 and 17.22 mmol·L ^?1 for S. costatum. All these results show that the effect of EMA on the growth of algae was species specific and mainly depended on IAD, which might be an important factor to influence algal growth.     

Investigations of carotenoids in some fauna of the Adriatic Sea. III. Leander (Palaemon) serratus and Nephrops norvegicus (Crustacea: Decapoda)

Investigations have been carried out on the carotenoids in Leander (Palaemon) serratus (Pennant) and Nephrops norvegicus (L.) (Crustacea: Decapoda) from the Adriatic Sea. The presence of the carotenoids was determined by means of columnar and thin-layer chromatography. The following carotenoids were found: Leander serratus: -carotene, echinenone, isocryptoxanthin, cryptoxanthin, canthaxanthin, lutein, isozeaxanthin, zeaxanthin, astaxanthin, and astacene; Nephrops norvegicus: -carotene, lutein, zeaxanthin, astaxanthin, and astacene.     

Comparative studies of carotenoids in the fauna of the Gullmar fjord (Bohuslän,Sweden). I.Alcyonium digitatum andPennatula phosphorea (Anthozoa)

Investigations have been carried out on the carotenoids in the anthozoan coelenteratesAlcyonium digitatum (yellow and brown specimens) andPennatula phosphorea from the Gullmar fjord (Bohuslän, Sweden). The presence of the carotenoids was determined by means of columnar and thin-layer chromatography. The following carotenoids were found inA. digitatum: canthaxanthin, phoenicoxanthin and phoeniconone, isozeaxanthin, zeaxanthin, astaxanthin, and astacene;P. phosphorea contained canthaxanthin, isozeaxanthin, zeaxanthin, astaxanthin, and astacene.     

Photosynthetic carboxylating enzymes in Phaeodactylum tricornutum: Assay methods and properties

Rapid freezing (in liquid nitrogen) of the marine diatom Phaeodactylum tricornutum Bohlin followed by thawing permits a convenient and sensitive measurement of the activities of carboxylating enzymes without the need to prepare a cell-free extract. Using this method, the properties of RuDP and PEP carboxylases have been compared with those assayed in cell-free extracts. The most significant difference was in the Michaelis' constants (K _m 's); the values being lower in the freeze/thaw assay. The absolute rate of carbon-dioxide fixation by the enzymes was less than the rate of photosynthesis by the intact alga. Significantly, the activity of PEP carboxylase was comparable (in some experiments, greater) to that of RuDP carboxylase. The significance of this and the possibility of an enzymatic approach to measurements of marine primary productivity are discussed.     

Growth and competition of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. II. Light limitation

The marine diatoms Phaeodactylum tricornutum (Bohlin) and Thalassiosira pseudonana (Hasle and Heimdal) were grown under both continous illumination and a 14 h light: 10 h dark cycle at light intensities ranging from 1.53×10^-4 to 2.95×10^-1 ly min^-1. Under both photoperiods, T. pseudonana exhibited higher division rates than P. tricornutum at high light intensities, but the reverse was true at all light intensities <3×10^-3 ly min^-1. Comparison of these results with available data on light-limited growth of other planktonic algae suggests that P. tricornutum may be unusually efficient at maintaining its cell division rate at low light intensity. This efficiency may contribute substantially to its success in turbid, nutrient-enriched mass algal culture systems, the only environments in which it is known to attain great numbers.Contribution No. 4086 from the Woods Hole Oceanographic Institution.     

Evaluation of the copepod Tigriopus californicus as a bioassay organism for the detection of chemical feeding deterrents produced by marine phytoplankton

Marine phytoplankton have been shown to use chemical feeding deterrents to reduce or inhibit zooplankton grazing. In order to screen phytoplankton species for feeding deterrent production and to isolate and identify feeding deterrent compounds, a new, rapid, and reliable laboratory bioassay was developed. This bioassay used the laboratory-reared harpacticoid copepod Tigriopus californicus and measured inhibition of feeding by measuring the fecal pellet production rate. The bioassay was capable of detecting deterrent compounds: (1) adsorbed onto ground fish food (a normally palatable food); (2) dissolved in a mixture of seawater and live Thalassiosira pseudonana cells (a species of diatom which had no feeding deterrent activity); and (3) present in live cell cultures. Method (2) was recommended for use in bioassay-guided fractionation (isolation of chemical compounds), as it was reliable, rapid, accurate, and easy to perform with large numbers of samples. The total bioassay time was < 48 h, and data collection required only a microscope. Methanolic cell extracts of several phytoplankton species were screened for feeding deterrent activity. Extracts from the diatom Phaeodactylum tricornutum and the dinoflagellate Gonyaulax grindleyi gave feeding deterrent responses, while extracts from the diatom Thalassiosira pseudonana gave no feeding deterrent responses. Live P. tricornutum cells deterred feeding at densities of 6x10⁵ cells ml^-1. This bioassay should provide a valuable tool in screening phytoplankton for feeding deterrent compounds and determining the chemical nature of these compounds.     

Questions on the mechanism of temperature adaptation in marine phytoplankton

The rate of light-saturated photosynthesis in 3 marine algae [Phaeodactylum tricornutum Bohlin, Nitzschia closterium (Ehrenberg) Smith and Dunaliella tertiolecta Butcher] varies during growth in batch culture. The photosynthetic rare declines most rapidly during growth at the higher temperatures. Because of these changes in photosynthesis rate, the previously reported enhanced photosynthetic abilities caused by growth at lower temperatures (generally interpreted as evidence for higher enzyme levels) can only be observed when measurements are made late in the exponential phase or after the onset of the stationary phase of growth. When allowance is made for the earlier peak of photosynthetic ability in cultures growing at higher temperatures, there is no evidence for adaptation to lower temperatures being caused by increased levels of the enzymes required for carbon-dioxide fixation. When the changes due to growth in batch culture are taken into account, certain effects of temperature can be recognized. the dry weight: chlorophyll ratio of all 3 algae increases with decreasing growth temperatures. For P. tricornutum and N. closterium, growth at lower temperatures reduces the cellular content of chlorophyll a, but has little effect on the chlorophyll content of D. tertiolecta. The dry weight: cell-number ratio of D. tertiolecta and P. tricornutum increases with lower growth temperatures, but growth temperature has little effect on the cell mass of N. closterium. Growth of the 3 algae at lower temperatures does not increase their ability to photosynthesize at these lower temperatures. Rather, it reduces their ability to assimilate carbon dioxide at the higher temperatures.     

A quantitative comparison of the commonly used methods for extracting carotenoids from avian plasma

Interest in animal carotenoids, especially in birds, has exploded in recent years, and so too have the methods employed to investigate the nature and function of these pigments. Perhaps the most easily and commonly performed procedure in this work has been the determination of carotenoid concentration from avian plasma. Over the past 20 years of research on avian carotenoids, numerous methods have been used to extract carotenoids from bird plasma, all of which have differed in several important parameters (e.g., number and types of solvents used, degree of mixing/centrifugation). However, to date, no study has systematically compared these methods to determine if any of them are more effective than others for recovering any or all types of carotenoids present. We undertook such an investigation on plasma samples from two bird species (house finch, Carpodacus mexicanus, and mallard, Anas platyrhynchos) using five of the most commonly employed methods for extracting carotenoids from avian plasma: (1) acetone-only, (2) methanol-only, (3) ethanol-only, (4) ethanol + hexane, and (5) ethanol + tert butyl methyl ether. We also manipulated the amount of time that extracts were centrifuged, which has varied tremendously in previous studies, to evaluate its importance on carotenoid recovery. We found that all methods equally recovered the polar xanthophylls (lutein and zeaxanthin), but that the methanol-only procedure poorly recovered non-polar carotenoids (less β-carotene in both species and less β-cryptoxanthin in house finches) compared to the other methods. These results suggest that the data accumulated to date on xanthophyll plasma carotenoids in birds should be comparable across studies and species despite the different chemical extraction methods used. However, care should be taken to use relatively strong organic solvents for fully recovering non-polar carotenoids. We also found no effect of centrifugation duration (1 vs. 10 min at 10,000 rpm) on carotenoid recoveries, demonstrating that researchers can save considerable time by centrifuging for a much shorter time period than is typically used.     

Feeding deterrence properties of apo-fucoxanthinoids from marine diatoms. II. Physiology of production of apo-fucoxanthinoids by the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana,and their feeding deterrent effects on the copepod Tigriopus californicus

The marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have been shown to produce apo-fucoxanthinoid compounds which act as feeding deterrents against the harpacticoid copepod Tigriopus californicus. The amounts and types of apo-fucoxanthinoids produced were species specific. Th. pseudonana produced small quantities of apo-12-fucoxanthinal and apo-13-fucoxanthinone only during senescence, while P. tricornutum produced much greater quantities of these two compounds during both log and senescence phases, in addition to producing a third compound, apo-10-fucoxanthinal, only during senescence. For both species, production of apo-fucoxanthinoids increased as the cells entered senescence phase due to phosphate limitation. The amounts of apo-fucoxanthinoids necessary to reduce feeding in T. californicus by 50% ranged from 2.22 to 20.2 ppm. This range was approximately 1000 times lower than the total apo-fucoxanthinoid concentration in P. tricornutum. The amounts of apo-fucoxanthinoids necessary to cause a 50% mortality in a population of T. californicus ranged from 36.8 to 76.7 ppm. Thus, these compounds are present in concentrations which may have ecological significance in the control of bloom formation and grazing. The production of apo-fucoxanthinoids may be a phenomenon common to many diatoms, particularly as they enter senescence due to nutrient limitation.     

The influence of suspension density and temperature on the filtration rate of Hiatella arctica

The rate of filtering Phaeodactylum tricornutum and Isochrysis galbana was measured in Hiatella arctica (L.) by the indirect suspension depletion method monitored by optical density measurement. The filtration rate of H. arctica was found to be 1.412×10^–2 l/h/g wet weight at a temperature of 15°C when fed with P. tricornutum, at average cell concentrations up to 3.5×10⁶ cells/ml. The filtration rate dropped almost to zero when the concentration of P. tricornutum reached 11×10⁶ cells/ml. The filtration rate of I. galbana diminished at a much lower cell concentration of 1×10⁶ cells/ml, and almost ceased at 3 to 4×10⁶ cells/ml. In mixed cultures of I. galbana and P. tricornutum, the filtration rate ratio was 0.37 to 1.00, and this was believed to be due to a proportion of the smaller former cells passing through the ostia. However, when resuspended in sea water, I. galbana cells were taken at a rate slightly less than P. tricornutum. The medium in which the I. galbana cells had been grown was inhibitory to the filtering activity of H. arctica, since, when cells of either alga were resuspended in the medium, the filtration rate was considerably reduced. No inhibitory factor existed in either of the original nutrient media. Hence, the importance of using low cell concentrations and of eliminating any inhibitory metabolic products when measuring filtration rates of bivalves is stressed. H. arctica shows a typical activity temperature eurve for a boreo-arctic species, with a steady rise from 0°C to a maximum between 15° and 17°C, and a sharp fall in activity to about zero at 25°C. The rates of filtration of various species at temperatures approaching the optimum were compared after allowance was made for fall in filtration rate with increasing body weight. The results suggested that the Mytilacea had the highest filtration rates and that H. arctica possesses one of the lowest filtration rates recorded.     

Carotenoid-based ornamentation as a dynamic but consistent individual trait

Carotenoid-based ornaments act as signals of quality in many animal species. In contrast to feathers, which are relatively stable structures, carotenoid-pigmented integuments (e.g. bills, lores, tarsi) can change colour rapidly and may better reflect changes in physiological condition. I studied the seasonal variations in plasma carotenoids in red-legged partridges (Alectoris rufa) kept on a constant diet and free of intestinal parasites. Furthermore, I analyzed whether seasonal changes in circulating carotenoids were mirrored by the carotenoid-based coloration of eye rings and bill of this species. Plasma carotenoids showed seasonal variation, with higher levels coinciding with the end of the mating and the start of the laying season. Eye ring pigmentation was related to plasma carotenoid levels, and changes in bill hue (but not changes in UV or red bill chroma) mirrored the variation in plasma carotenoids during the breeding season. Despite the seasonal variation, individual differences in eye ring pigmentation and bill hue, UV and red chroma were consistent throughout the breeding season. Similarly, individual differences in eye ring pigmentation and bill hue and red chroma remained consistent between consecutive years. These results suggest that carotenoid based integumentary colorations act as dynamic traits that accurately reflect the carotenoid-status of individuals, thus reliably indicating consistent differences in individual quality. Furthermore, variability in signal expression appears to have a relevant genetic/phenotypic basis independently of environmental conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Presence of β-carotene and a number of xanthophylls in Hyale perieri (Crustacea: Amphipoda) from the Black Sea

Investigations have been made on the carotenoids in the marine amphipod Hyale perieri (Lucas) from the Black Sea. The presence of the carotenoids -carotene, canthaxanthin, lutein, neoxanthin and astaxanthin was determined by means of columnar and thin-layer chromatography. It is highly probable that violaxanthin is also present in H. perieri.     

Investigations of carotenoids in some fauna of the Adriatic Sea. I. Verongia aerophoba (Porifera: Spongiidae)

Investigations have been carried out on the carotenoids in Verongia aerophoba (Schmidt) (Porifera: Spongiidae) from the Adriatic Sea. The presence of the carotenoids -carotene, -carotene, isozeaxanthin, lutein (ester and free), zeaxanthin, violaxanthin, neoxanthin, astaxanthin and astacene was determined by means of columnar and thin-layer chromatography.     

Relative growth of different species of marine algae in wasterwater-seawater mixtures

In recent studies, we developed a combined nutrient removal-marine aquaculture process for the tertiary treatment of wastewater and the production of commercially important shellfish. Part of this process consists of an outdoor mass cultivation system for marine algae. During our previous experiments we observed that marine diatoms almost exclusively are the dominant algal species in our outdoor cultures. To better understand this phenomenon of diatom dominance we grew 16 species of marine algae in continuous monoculture under laboratory conditions simulating to some degree the conditions prevailing in our outdoor experiments. Species such as Skeletonema costatum, Monochrysis lutheri and Tetraselmis sp., which were never dominant in our outdoor cultures, grew as well in monoculture, as Phaeodactylum tricornutum, frequently, the prevalent species outdoors. However, when monocultures of Dunaliella tertiolecta and Thalassiosira pseudonana (3H) were purposely contaminated with P. tricornutum, the latter species quickly became dominant. It is suggested that a complex interaction of environmental factors is usually responsible for the dominance of a particular species; one such factor may be the nitrogen source in the growth media. Under conditions of virtually, complete nitrogen assimilation, the carbon: nitrogen ratio in the algae was high (7 to 8) when NO ₃ ^- –N was the source of nitrogen, and low (4 to 6) when NH ₄ ⁺ –N was the prime form of nitrogen. When algal growth was low, resulting in a large inorganic nitrogen residue, the carbon:nitrogen ratio was low regardless of whether NO ₃ ^- –N or NH ₄ ⁺ –N was the main nitrogen source.Contribution No. 3297 from the Woods Hole Oceanographic Institution.     

Biosynthetically active glutamine synthetase in the marine diatom Phaeodactylum tricornutum: optimization of the forward-reaction assay

The activity of glutamine synthetase (GS) was measured in the marine diatom Phaeodactylum tricornutum Bohlin (Strain SME) by a biosynthetic assay, based on -glutamyl hydroxamate synthesis, and referred to as the forwardreaction assay. The effects of pH, temperature and different homogenizing buffer preparations on enzyme activity, linearity of reactions, and substrate-saturation kinetics were investigated. The resultant data provide the basis for establishing optimum experimental conditions for a standard assay. Affinities of P. tricornutum GS for glutamate, ATP and Mg²⁺ were similar to those recorded elsewhere for a variety of other phytoplankton species using true biosynthetic assays based on release of inorganic phosphate, whereas the affinity for hydroxylamine was two orders of magnitude lower than that for ammonium, with an apparent K _m value in the millimolar range. This, together with negative results obtained during earlier attempts to detect GS activity in P. tricornutum using the true biosynthetic assay, indicates that the GS of this alga has a lower affinity for ammonium than that of other phytoplankton species. Dual substrate kinetics demonstrated that apparent K _m and V _m values for glutamate were directly proportional to the concentration of ATP, thus giving indirect evidence of a correlation between GS activity and the adenylate energy charge. Comparisons between synthetase activities obtained with the optimized forward-reaction assay and transferase activities reported from other studies on various phytoplankton species revealed discrepancies which, to a great extent, probably arise from differences in the growth conditions of the organisms.     

Pigments of the Porifera: Demospongiae. I. Carotenoids of Axinella verrucosa

Studies were made of some carotenoids extracted from Axinella verrucosa (O. schmidt) (Porifera: Demospongiae), a species which occurs in the Gulf of Naples. -carotene, renieratene, isorenieratene, and an unknown isomer of renieratene have been isolated.     

Bioseston production by Mytilus edulis and its effect in experimental systems

Mytilus edulis L. (Bivalvia: Mollusca) can produce appreciable quantities of 3.4 to 5.4 m diameter bioseston, when previously fed on Phaeodactylum tricornutum and Tetraselmis suecica. Bioseston production, observed in flowing filtered sea water, is shown to originate from resuspension of faeces and can cause filtration-rate underestimates of up to 35% under certain experimental conditions. Feeding with Thalassiosira pseudonana below a concentration of 4x10⁷ particles l^-1 caused no significant bioseston production and this alga would, therefore, be suitable for use in quantitative estimates of filtration rate.