Preparative and analytical extraction of pigments from brown algae with dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) rapidly extracts much of the chlorophyll c and fucoxanthin from the intact thalli of brown algae. Subsequent extraction by acetone rapidly removes most of the chlorophyll a and -carotene. An analytical procedure for these pigments has been developed, based upon this extraction sequence, followed by partition of the acetone extract between hexane and aqueous acetone. Fucoxanthin and chlorophyll c can be recovered from the DMSO extract and the aqueous acetone phase by extraction into ethyl acetate and subsequent evaporation. The DMSO-acetone extraction sequence is also effective with green algae and a few red algae, although the full scope of the method has not been investigated.     

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.     

Carotenoids in the hermit crab Clibanarius misanthropus (Crustacea,Paguridae) from the Black Sea

The authors investigated the carotenoids in the hermit crab, Clibanarius misanthropus (Risso), from the Black Sea. By means of columnar and thin-layer chromatography the following carotenoids were found to be present in that species; -carotene, canthaxanthin, lutein, astaxanthin and some kind of polioxyxanthophill.     

Some carotenoids in the jelly-fish Aurelia aurita (Scyphozoa: Discomedusae)

The author investigated the carotenoids in the jelly-fish Aurelia aurita (L.), from the Baltic Sea. By means of columnar and thin-layer chromatography, the following carotenoids were found to be present in that species: -carotene, echinenone isocryptoxanthin, canthaxanthin, lulein, zeaxanthin, astaxanthin and astacene.     

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.     

Profiles of photosynthetic pigments in the ocean using thin-layer chromatography

Phytoplankton pigments at a coastal station off Sydney, Australia, were studied by cellulose thinlayer chromatography. The chromatographic procedure distinguished chlorophylls a, b and c, chlorophyllide a, pheophytin a and pheophorbide a, and the major carotenoids carotene, astaxanthin, fucoxanthin, peridinin, diadinoxanthin and neoxanthin. Chlorophyllide a and pheophorbide a were the most significant chlorophyll-a degradation products in the water column, chlorophyllide a coming from chlorophyllase activity of senescent diatoms, and pheophorbide a from faecal pellets of copepods. Chlorophyll c occurred in every sample, even where there was no trace of chlorophyll a. Because of the greater chemical and biological stability of chlorophyll c compared to chlorophyll a, high chlorophyll c:a ratios result from a large proportion of senescent or detrital material in the samples. Determining the position of patches of copepods, diatoms, green algae and dinoflagellates in the water column was easily done by noting the presence of definitive pigments on the chromatograms.     

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.     

Carotenoids in the tiger prawn Penaeus esculentus during ovarian maturation

Female Penaeus esculentus Haswell were collected by 15 to 20 min duration trawls during 1990. Carotenoids were analysed in the digestive gland, abdominal muscle, the remainder of the body (hereafter called integument) and ovary of prawns in Stage 2 through Stage 4 (fully mature) of maturation. The only oxycarotenoids (xanthophylls) identified were astaxanthins or astaxanthin esters; occasionally low levels of -carotene were detected in the digestive gland. The concentrations of astaxanthin monoesters (AM) and diesters (AD) were highest, with only minor amounts of free astaxanthins (Ast), except in the maturing ovaries, where free astaxanthins predominated (up to 80% of the total carotenoid). Of the total carotenoid, 82 to 94% was in the integument, but at maturity the digestive gland contained 10.7±3.4% and the ovary 5.6±0.9% of the total carotenoid. Only the ovary increased in mass during maturation, reaching up to 5.2% of total prawn mass. During this period, digestive gland concentrations of AM, AD and Ast all increased (tota 20 to 120 g g^-1); levels in the muscle and integument varied little throughout maturation (total 0.4 and 100 g g^-1, respectively); ovary AM levels remained low throughout (1.5 to 1.2 g g^-1), AD increased from only 2 to 5 g g^-1, but Ast increased from 2 to 34 g g^-1. Apart from the ovary, AM concentrations were the most variable. In common with other decapod Crustacea, the maturing ovary of P. esculentus contained high levels of carotenoids, indicating that these may have an important role in early development. The natural diet of P. esculentus includes a variety of carotenoids, but except for a little -carotene, the digestive gland, where absorption occurs, contained astaxanthins, with only an occasional trace of -carotene. This suggests that the conversion of dietary carotenoids to astaxanthin occurs soon after ingestion.     

Ecotypic differentiation in the marine diatom Skeletonema costatum: influence of light intensity on the photosynthetic apparatus

Three genetically distinct clones of Skeletonema costatum (Grev.) Cleve were grown at 20°C under high (274 E m^-2 s^-1) and low (27 E m^-2 s^-1) light conditions and their photoadaptive photosynthetic responses compared. When all three clones were grown under low light, pigment analyses and fluorescence excitation spectra demonstrated that the accessory pigments, chlorophyll c and fucoxanthin, became more important in light-harvesting compared to chlorophyll a. Photosynthetic unit sizes increased for Photosystems I and II in low light, but photosynthesis vs irradiance characteristics were not reliable predictors of photosynthetic unit features. Fluorescence excitation spectra and photosynthesis vs irradiance (P-I) relationships indicated that changes in energy transfer occurred independent of changes in pigment content. Large increases in accessory pigment content were not accompanied by large increases in excitation from these pigments. Changes in energy transfer properties were as important as changes in PSU size in governing the photoadaptive responses of S. costatum. When the three clones were grown under identical conditions, each had a separate and distinct pattern of photoadaptation. Significant differences among clones were found for pigment ratios, photosynthetic unit sizes for Photosystems I and II and efficiency of energy transfer between pigments. These strikingly different photoadaptive strategies among clones may partially account for the great ecological success of the diatom species. This is the first quantitative investigation of the importance of both chlorophyll c and fucoxanthin to the adaptive responses of diatoms to light intensity, and represents the most complete characterization of the photoadaptive responses of a single species of marine phytoplankter to differences in light environment.     

Microzooplankton grazing and selectivity of phytoplankton in coastal waters

Microzooplankton grazing activity in the Celtic Sea and Carmarthen Bay in summer 1983 and autumn 1984 was investigated by applying a dilution technique to high-performance liquid chromatographic (HPLC) analysis of photosynthetic pigments in phytoplankton present within natural microplankton communities. Specific grazing rates on phytoplankton, as measured by the utilisation of chlorophyll a, were high and varied seasonally. In surface waters during the autumn, grazing varied between 0.4 d^-1 in the bay and 1.0 d^-1 in the Celtic Sea, indicating that 30 and 65% of the algal standing stocks, respectively, were grazed daily. Grazing rates by microzooplankton within the thermocline in summer suggest that 13 to 42% of the crop was grazed each day. Microzooplankton showed selection for algae containing chlorophyll b, in spite of a predominance of chlorophyll c within the phytoplankton community. Changes in taxon-specific carotenoids indicated strong selection for peridinin, lutein and alloxanthin and selection against fucoxanthin and diadinoxanthin. This indicates a trophic preference by microzooplankton for dinoflagellates, cryptophytes, chlorophytes and prasinophytes and selection against diatoms, even when the latter group forms the largest crop within the phytoplankton. Interestingly, those algal taxa preferentially grazed also showed the highest specific growth-rates, suggesting a dynamic feed-back between microzooplankton and phytoplankton. Conversion of grazing rates on each pigment into chlorophyll a equivalents suggests firstly, that in only one experiment could all the grazed chlorophyll a be accounted for by the attrition of other chlorophylls and carotenoids, and secondly that in spite of negative selection, a greater mass of diatoms could be grazed by microzooplankton than any other algal taxon. The former may be due either to a fundamental difference in the break-down rates of chlorophyll a compared to other pigments, or to cyanobacteria forming a significant food source for microzooplankton. In either case, chlorophyll a is considered to be a good measure of grazing activity by microzooplankton.     

β-glucanase activity in the seastar Pisaster ochraceus

Specimens of Pisaster ochraceus, an intertidal carnivorous seastar, were collected in northern Puget Sound at Post Point, near Bellingham, Washington, USA, in November 1993, and used in experimentation through September 1994. Ammonium sulfate fractions (60% precipitates) from excised pyloric caeca (digestive glands) possessed -glucanase activity directed toward such -linked substrates as carboxymethylcellulose (CMC), partially-digested cellulose (cellodextrins) and the synthetic substrate p-nitrophenyl--D-glucoside (PNDG). pH-activity and pH-stability maxima were at pH 6 and 5, respectively. No hydrolytic activity was detectable on native cellulose, native chitin or on cationic-modified celluloses such as DEAE-cellulose. Gel filtration (Sephadex G-100) of this 60% precipitate followed by DEAE-Sephadex column chromatography revealed heterogeneity of -glucanase activity, with an indication of up to 11 distinct fractions with variable activity directed toward the substrates indicated above (CMC, cellodextrins and PNDG). These fractions displayed differing ratios of hydrolytic activity on these substrates. None of the isolated fractions displayed activity toward either native cellulose or native chitin. Gut extracts from the edible mussel (Mytilus edulis), a common prey species of P. ochraceus, were shown to possess partially digested cellulose (cellodextrins). These cellodextrins were further degraded by seastar -glucanases in vitro. The digestion of the gut contents of ingested prey provides a possible adaptive explanation for the presence of -glucanase activity in a carnivore.     

Pigment content and photosynthetic rate of the fronds of Macrocystis pyrifera

The Macrocystis pyrifera (L.) C. Ag. frond is here described in terms of chlorophyll a, fucoxanthin, chlorophyll c and photosynthetic rate. Pigment concentrations increased back from the apical meristem reaching a maximum after 2 to 3 m. Pigment concentrations were then generally constant throughout most of the length of the frond, finally decreasing again in the oldest parts of the frond with the exception of the sporophylls. Pigment ratios remained relatively constant throughout. Maximum net photosynthetic rates on a given frond showed a decrease with tissue age on both an area basis (1040 down to 463 nmol O₂ cm^-2 h^-1) and on a chlorophyll a basis, which was shown as half-saturation constants (quantum irradiance) which dropped on an area basis from 85 mol m^-2 sec^-1 at 4.5 m above the holdfast to 26 mol m^-2 sec^-1 at 15.5 m. Young sporophytes transplanted from the sea floor to the surface (12 m) tended to decrease pigment content, while those transplanted to the bottom tended to increase all pigments, but especially fucoxanthin. Photosynthetic rates, however, changed little on a unit area basis. The results of these data are considered in the light of recent work on photosynthetic units, tissue age effects and general adaptations of the M. pyrifera frond to its light environment.     

Carbon and nitrogen dynamics during growth and degradation of phytoplankton under natural surface irradiance

Under conditions of natural irradiance, the development and decline of a flagellate-dominated phytoplankton population was followed in a coastal North Atlantic pond over a 3 d period in summer 1986. Irradiance negatively affected phytoplankton biomass estimated as chlorophyll a, which decreased during the day at photosynthetically available radiation (PAR) levels above 600 to 1000 mol m^-2s^-1; chlorophyll a increased at PAR values below this threshold. In addition, an inverse relationship was found between changes in chlorophyll a and changes in dissolved inorganic nitrogen, indicating synthesis of nitrogenous biomass mainly at night and degradation mainly during the day, with intense exchanges of material between the particulate and dissolved nitrogen fractions. The natural abundance of ¹³C in particulate matter increased initially, and then remained constant, and was controlled mainly by the ratio -carboxylases activity: ribulose biphosphate carboxylase activity. The hypothesis that the latter enzyme is broken down under high irradiance and is partly responsible for increases in external dissolved nitrogen was rejected.     

Studies of the pigments in the shrimp Crangon crangon (Crustacea: Malacostraca)

The material examined consisted of adult specimens (male and female) of the shrimp Crangon crangon (L.) collected during the summer of 1967 from the Gdask Bay of the Baltic Sea. Column and thin-layer chromatography was used for the separation of the carotenoids. The ester compounds and protein complexes of carotenoids were hydrolized with 15% KOH in methanol at room temperature in a nitrogen atmosphere for 48 h. The absorption maxima of the various fractions and spots were determined on the spectrophotometer Specol after eluation. The identification of the carotenoids was based mainly on the absorption maxima in appropriate organic solvents and on the R_t values. The acetone extract from the Crangon crangon specimens gave a light blue fluorescence and absorption maxima at wavelengths of 447, 463, 595 to 596 and 667 m. The following carotenoids were found to be present in the adult specimens of Crangon crangon: -carotene, cantaxanthin, astaxanthin, free and esterified lutein, zeaxanthin, and a form of xanthophyll which was not precisely identified. In addition to the above, biliary dyes were also found, probably of the mesobiliviolin and mesobilirhodin type.     

A comparative study of photoadaptation in four diatoms isolated as endosymbionts from larger foraminifera

Four endosymbiotic diatoms were isolated from 2 species of larger foraminifera collected in the Red Sea and Hawaii. The photoadaptive responses of the cultured diatoms were measured at 312, 19 and 7 W cm^-2. Two of the diatoms (Fragilaria shiloi and Nitzschia laevis), both isolated from Amphistegina lessonii, grew fastest at 312 W cm^-2. The other two diatoms (N. valdestriata and N. panduriformis) which were isolated from Heterostegina depressa, grew best at 19 W cm^-2. Of the four diatoms, F. shiloi grew best at high light levels. Also in F. shiloi, chlorophyll c content per cell was directly proportional to light intensity; in contrast chlorophyll a and carotenoids increased to maxima at 19 W cm^-2. The chlorophyll a and c and carotenoid content of N. valdestriata were also maximal at 19 W cm^-2. Photosynthetic rates, measured by respirometry, suggested that the diatoms were photoinhibited at higher light intensities and did well at moderately low light intensities (175W cm^-2). The photocompensation points of all 4 diatoms were about 2% of the light available in the spring at 1-m depth at Elat on the Red Sea. At Elat the photocompensation point would lie between 40 and 50 m if the algae were free in nature. The amount of attenuation of light by the shells of the host has not yet been measured. Presumably photocompensation of the algae within hosts is reached at depths less than 40 m.     

Seasonal study of phytoplankton pigments and species at a coastal station off Sydney: Importance of diatoms and the nanoplankton

Phytoplankton pigments and species were studied at a coastal station off Sydney (New South Wales, Australia) over one annual cycle. Sudden increases in chlorophyll a (up to 280 mg m^-2), due to short-lived diatom blooms, were found in May, July, September, January and February. These were superimposed upon background levels of chlorophyll a (20 to 50 mg m^-2), due mostly to nanoplankton flagellates, which occurred throughout the year. The nanoplankton (<15 m) accounted for 50 to 80% of the total phytoplankton chlorophyll, except when the diatom peaks occurred (10 to 20%). The annual cycle of populations of 16 dominant species-groups was followed. Possible explanations as to alternation of diatom-dominated and nanoplankton-dominated floras are discussed. Thin-layer chromatography of phytoplankton pigments was used to determine the distribution of algal types, grazing activity, and phytoplankton senescence in the water column. Chlorophyll c and fucoxanthin (diatoms and coccolithophorids) and chlorophyll b (green flagellates) were the major accessory pigments throughout the year, with peridinin (photosynthetic dinoflagellates) being less important. Grazing activity by salps and copepods was apparent from the abundance of the chlorophyll degradation products pheophytin a (20 to 45% of the total chlorophyll a) and pheophorbide a (10 to 30%). Chlorophyllide a (20 to 45%) was associated with blooms of Skeletonema costatum and Chaetoceros spp. Small amounts of other unidentified chlorophyll a derivatives (5 to 20%) were frequently observed.     

Effects of nitrogen and phosphorus enrichement on in vivo symbiotic zooxanthellae of Pocillopora damicornis

The reef coral Pocillopora damicornis (Linnaeus) was grown for 8 wk in four nutrient treatments: control, consisting of ambient, unfiltered Kaneohe Bay seawater [dissolved inorganic nitrogen (DIN, 1.0 M) and dissolved inorganic phosphate (DIP, 0.3 M)]; nitrogen enrichment (15 M DIN as ammonium); phosphorus enrichment (1.2 M DIP as inorganic phosphate); and 15 M DIN+1.2 M DIP. Analyses of zooxanthellae for C, N, P and chlorophyll a after the 8 wk experiment indicated that DIN enrichment increased the cellular chlorophyll a and excess nitrogen fraction of the algae, but did not affect C cell^-1. DIP enrichment decreased both C and P cell^-1, but the decrease was proportionally less for C cell^-1. the response of cellular P to both DIN and DIP enrichment appeared to be in the same direction and could not be explained as a primary effect of external nutrient enrichment. The observed response of cellular P might be a consequence of in situ CO₂ limitation. DIN enrichment could increase the CO₂ (aq) demand by increasing the net production per unit area. DIP enrichment could slow down calcification, thus decreasing the availability of CO₂ (aq) in the coral tissue.Hawaii Institute of Marine Biology Contribution No. 920     

Floristic and physiological differences between the shallow and the deep nanophytoplankton community in the euphotic zone of the open tropical Atlantic revealed by HPLC analysis of pigments

Suspended matter sampled in 1982 in the North Equatorial Current, in the open Atlantic to the west of West Africa, was analyzed by high performance liquid chromatography. The pigment fingerprint of samples taken in the surface mixed layer was dominated by zeaxanthin and chlorophyll a, in agreement with observed dominance of coccoid cyanobacteria. Near the bottom of the euphotic zone the fingerprint was more complicated, with a sharp transition at the depth of the deep chlorophyll maximum layer to dominance of chlorophyll b, 19-hexanoyloxyfucoxanthin and an unknown fucoxanthin derivative in the lower part of this layer; this fingerprint suggests dominance of eukaryotes (green algae, Prymnesiophyceae and Chrysophyceae) at depth. Up to 90% of the chl a was contained in particles smaller than 8 m, and in the surface mixed layer even more than 50% in particles smaller than 1 m. The high concentration of zeaxanthin relative to chl a near the surface suggests adaptation of the cyanobacteria to exposure to high irradiance. Evidence of this adaptation was the very high specific phytoplankton growth rate between sunrise and sunset (=0.16 h^-1), measured by recording ¹⁴C incorporation into organic carbon and into chl a carbon after isolation of the latter by HPLC. The high concentration of chl b relative to chl a at depth was possibly caused by shade-adapted green algae containing more chl b than chl a. The specific growth rate of the deep shade community was low (<0.04 h^-1), yet net primary production, calculated on the basis of chl a increase during incubation, was greatest at depth.     

Effect of UV-B radiation on the marine diatoms Lauderia annulata and Thalassiorsira rotula grown in different salinities

The marine diatoms Lauderia annulata Cleve and Thalassiosira rotula Meunier were grown at different salinities (20, 35 and 45) and exposed to different levels of midultraviolet, UV-B) 439, 717 and 1230 J m^-2 d^-1, weighted) for 2 d. A low UV-B dose (439 J m^-2 d^-1) usually caused a slight increase in biomass production (dry weight) compared to non-UV-B irradiated cells. Enhanced UV-B radiation (717 J m^-2 d^-1) depressed protein and pigment content (chlorophyll a, chlorophyll c ₁+c₂ and carotenoids), especially in algae grown at 20 or 35 salt concentration of the nutrient solution. The effect of UV-B radiation (717 J m^-2 d^-1) on the pattern and concentration of amino acids was species-dependent. Aspartic acid was reduced in all tested diatoms. A drastic increase in glutamine and a reduction in glutamic acid pools could be observed in L. annulata samples, but no significant variation of the impact of UV-B was found in dependence on the salt concentration of the nutrient medium. T. rotula cells grown at 35 S showed an increase of glutamic acid and a decrease of glutamine levels after UV-B radiation. The results are discussed in relation to the impact of UV-B upon carbon and nitrogen metabolism.     

Transformation of progesterone by marine bacteria

The progesterone transforming ability of marine bacteria has been determined. 20 of the 53 marine cultures tested were found capable of transforming progesterone to 6-hydroxyprogesterone and/or to 11,17-dihydroxyprogesterone.