Marine Ecology Progress Series 324:113

ABSTRACT: Phospholipid-derived fatty acids (PLFA) are widely used as chemotaxonomic markers in microbial ecology. In this paper we explore the use of PLFA as chemotaxonomic markers for phytoplankton species. The PLFA composition was determined for 23 species relevant to estuarine phytoplankton. The taxonomic groups investigated were Chlorophyceae, Trebouxiophyceae, Prasinophyceae, Bacillariophyceae, Dinophyceae, Cryptophyceae, Rhodophyceae, Pavlovophyceae and Prymnesiophyceae. Most of these taxonomic groups have a characteristic PLFA composition, although a few contain truly unique PLFA biomarkers. The PLFA composition was used to derive species composition and abundance of the phytoplankton in the Scheldt estuary (border region between Belgium and the Netherlands) in April, July and October 2003, using the matrix factorization program CHEMTAX. The results agree well with results from HPLC-derived pigment analysis of the same samples. The advantages of using PLFA instead of pigments, or rather in addition to pigments, are that (1) information on bacteria can be derived in addition to information on photosynthetic taxa, (2) in some cases more or different taxa can be distinguished and (3) the isotopic composition of PLFA can be measured relatively easily, allowing the measurement of group-specific primary production. KEY WORDS: Phytoplankton composition · Phospholipid-derived fatty acids · PLFA · Scheldt estuary · Biomarkers · CHEMTAX Resale or republication not permitted without written consent of the publisher Mar Ecol Prog Ser 324: [113][114][115][116][117][118][119][120][121][122][123][124][125] 2006 microscopic enumerations require a trained microscopist and are time-consuming. Furthermore, in most cases, the algae have to be stored and therefore fixed before analysis and not all taxa withstand fixation well The use of chemotaxonomic markers provides an alternative method for determining phytoplankton composition. Photosynthetic pigments are the best known chemotaxonomic markers for phytoplankton. As HPLC technology advances, more and more pigments can be routinely measured, allowing an increasing number of taxa to be distinguished (e.g. Bacteria are also an important constituent of aquatic ecosystems. Suitable pigment biomarkers are lacking for bacteria, and phospholipid-derived fatty acids (PLFA) have become well-established chemotaxonomic markers The objective of the present study was to elaborate and refine the use of PLFA to determine phytoplankton composition and abundance in natural samples. We have determined the PLFA composition of a number of phytoplankton species relevant to estuarine phytoplankton and used these data to derive group composition and abundance in the Scheldt estuary (border region between Belgium and the Netherlands) from the PLFA composition of particulate suspended matter. The results from this PLFA analysis are compared to similar data derived from the pigment composition of the same samples. In a later paper we will determine the photosynthetic activity of several taxonomic groups by measuring the rate of 13 C-labeling of individual PLFA (Dijkman et al. unpubl.). MATERIALS AND METHODS Organisms and culture conditions. PLFA composition was determined for a number of species from taxonomic groups relevant to estuarine phytoplankton. Most of these species are regularly observed in the Scheldt estuary (e.g. Study area and sampling. Field measurements were done in the Scheldt estuary, a well-mixed tidal system in the border region between Belgium and The Netherlands Sample treatment. Samples from cultured material were filtered onto precombusted (400 o C) GF/F filters (Whatman); field samples were filtered onto precombusted GF6 filters (Schleicher & Schuell). Samples were frozen immediately in liquid nitrogen. Samples for pigment analysis were stored at -80°C until extraction; PLFA extraction was started immediately. Samples for 114 Dijkman & Kromkamp: PLFA as phytoplankton biomarker microscopic analysis were preserved with 2% formaldehyde and stored in the dark at 5°C. Filters for the analysis of particulate organic carbon (POC) content were stored at -20°C and were dried before determination of the POC content using an elemental analyzer. PLFA extraction. Total lipids were extracted in a mixture of chloroform, methanol and water (1:2:0.8 by vol.) according to Boschker (2004), using an adaptation of the method of Bligh & Dyer (1959). Phase separation was induced by the addition of chloroform and water to a final composition of chloroform:methanol:water of 1:1:0.9 (by vol.). The chloroform layer containing the total lipid fraction was collected. This total lipid fraction was fractionated into neutral-, glyco-and phospholipids on heat-activated silicic acid columns (60, Merck ) by sequential elution with chloroform, acetone and methanol. The methanol contained the most polar fraction, which consisted mainly of phospholipids. This fraction was derivatized using mild alkaline methanolysis to yield fatty acid methyl esters (FAME). 12:0 and 19:0 were used as internal FAME standards. FAME concentrations were determined by gas chromatography-flame ionization detection (GC-FID, Interscience HRGC MEGA 2 series) using a polar analytical column (Scientific Glass Engineering BPX-70). Identification was based on comparison of retention times with known reference standards and by gas chromatographymass spectrometry (Finnigan Voyager Mass Spectrometer). Fatty acid notation. Fatty acid notation consists of the number of carbon atoms followed by a colon and the number of double bonds present in the molecule. The position of the first double bond relative to the aliphatic end of the molecule follows the symbol omega, ω (e.g. 16:1ω7). The other possible double bonds are methylene interrupted. The prefixes 'i' (iso) and 'ai' (anteiso) represent the location of a methyl branch 1 and 2 carbons, respectively, from the aliphatic end (e.g. i15:0). Where relevant, 'c' indicates the cis-orientation of the double bonds. Pigment analysis. Pigments were extracted in 90% acetone. Filters and cells were disrupted by shaking with glass pearls in a bead beater, which was cooled using CO 2 gas. The slurry was centrifuged and the pigment content of the supernatant was analyzed by HPLC. Pigments were separated on a reverse-phase column (Nova-pak C18; Waters Millennium HPLC system). Gradient mixing pumps delivered 3 mobilephase solvents: methanol/ammonium acetate, 90% acetonitril and 100% ethyl acetate (Rijstenbil 2003 and references therein). Pigments were identified and quantified by commercially available standards using both a fluorescence detector (Waters 474 Scanning Fluorescence Detector) and a photodiode array absorption detector (Waters 996 Photodiode Array Detector). Data analysis. Data were analyzed using the matrixfactorization program CHEMTAX RESULTS PLFA composition of algal cultures The PLFA composition of each species was determined in duplicate. Average values are reported in In all tested species, 16:0, 16:1ω7c, 18:1ω9c and 18:2ω6c were present, although the relative abundance varied considerably and the latter 2 were present in the Bacillariophyceae in small amounts only. Interesting and useful differences occurred in the presence and relative abundance of 14:0, poly-unsaturated fatty acids (PUFA) with a chain length of 16 (16PUFA) and 18 (18PUFA) carbon atoms, 20:4ω6, 20:5ω3 and 22:6ω3. The Chlorophyceae and the Trebouxiophyceae differed from the other taxa by lacking PUFA longer than 18 carbon atoms and having no or only negligible amounts of 14:0. Both taxa had a small contribution of 16:2ω6 and a large fraction of 18:3ω3. The Trebouxiophyceae contained a considerable fraction of 16:3ω3 but lacked 16:4ω3 and 18:4ω3. Of the Trebouxiophyceae tested, 1 (Chlorella autotrophica), contained 20:0. 16:3ω3 was only a minor fraction in the Chlorophyceae, whereas it was a major component in the Trebouxiophyceae. Instead, 16:4ω3 and 18:4ω3 were present as important fractions in the Chlorophyceae. The PLFA content of the Prasinophyceae was similar to that of the Chlorophyceae, but contained in addition the long fatty acids 22:5ω3 and 22:6ω3. The 18:3ω3 fraction was only 30 to 50% of that found in the 2 other Chlorophyte taxa. The Bacillariophyceae were characterized by a relatively large fraction of 14:0 and the occurrence of 16:2ω7, 16:2ω4, 16:3ω4, 16:4ω1, 20:5ω3 and 22:6ω3. 18:4ω3 comprises only a minor fraction in most Bacillariophyceae and the contribution of 18:3ω3 was less than 1% in all species tested. The Cryptophyceae contain only PLFA that are also found in either the Chlorophyta (Chlorophyceae, Trebouxiophyceae and Prasinophyceae) or the Bacillariophyceae. Important PLFA are 18:3ω3, 18:4ω3, 20:5ω3 and 22:6ω3, whereas 14:0 is found in low amounts. In the Rhodophyceae 20:4ω6 was a major fraction; 20:5ω3 also made up an important fraction of the PLFA in this group and 16PUFA were lacking. The Dinophyceae had a PLFA composition similar to that of the Cryptophyceae. Remarkable is the low contribution of 16:1ω7c and of 20:5ω3 in Heterocapsa triquetra, 1 of the 2 tested species. The Dinophyceae contain 2 PLFA that were not found in any of the taxa described above, namely 18:5ω3 and an unidentified PUFA with a chain length of 20 carbon atoms. The chain length was deduced from retention time, mass spectra indicated that this PLFA is a PUFA. This PLFA is designated 20:xωx In the Haptophyta, 2 groups seemed to be present, with one group resembling the Dinophyceae and the other more closely resembling the Bacillariophyceae. In all 4 species examined, 14:0, 18:4ω3 and 22:6ω3 were found; 18:5ω3 and the same 20:xωx as found in the Dinophyceae were present in Emiliania huxleyi and Phaeocystis globosa, but absent in Pavlova lutheri and Isochrysis sp.. The latter 2 species contained a high percentage of 20:5ω3, whereas this was present only in a very low amount in the former 2 species. In contrast to the Bacillariophyceae, Haptophyta did not contain 16PUFA. These 2 groups in the Haptophyta will be designated Haptophyta I (E. huxleyi and P. globosa) and Haptophyta II (P. lutheri and Isochrysis sp.) respectively. Values are the average of 2 measurements. Some PLFA that were found at a very low concentration or occurred in a single species only were omitted from table. 20:xωx is an unidentified PUFA with a chain length of 20 carbon atoms. PLFA are grouped into saturated fatty acids (SAFA), mono-unsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). Within each group, PLFA are listed according to GC retention time. Species abbreviations as in SAFA Pigment composition of algal cultures The pigment composition of the species for which the PLFA composition is described above was measured, but is not discussed in detail here. The results agreed well with general data on the pigment composition of microalgae. Remarkable was that our strain of Phaeocystis globosa contained only a minute amount of 19'-hexanoyloxyfucoxanthin, a pigment which is often used as a biomarker for this species. PLFA and pigment in the Scheldt Estuary The variations in PLFA and pigment concentration and composition are shown for July 2003 in Individual PLFA concentrations are expressed as a percentage of total PLFA in Mar Ecol Prog Ser 324: 113 Group abundance The matrix factorization program CHEMTAX was used to estimate group abundance from the pigment and PLFA composition. Pigment and PLFA data in combination with microscopic observations were used to decide which taxa should be added to the input ratio matrix. The input ratio matrices for both pigment and PLFA data are shown in Relative group abundance estimated from the pigment data is shown as the contribution of each group to chl a in CHEMTAX adapts the input ratio file (within limits set by the user) to best fit the field data. The adapted PLFA ratio file of 28 PSU is shown in The outcome of the CHEMTAX analysis changed little when non-unique PLFA like 18:1ω9c were left out or when PLFA present only in small fractions like 16:2ω7 or 16:2ω6 were added. We chose to analyze the data per station, since this caused the ratio file to be adapted such that the variations observed in the estuary in 16:2ω4, 16:3ω4 and 16:4ω1 were reflected in the adapted ratio file. However, when all the data were analyzed in a single file instead of dividing the data per station, this had little effect on the outcome. The only group often affected by different fitting options was the Cryptophyceae, probably because this group lacks unique biomarkers. The choice of the PLFA composition of marine sediments to define bacteria is arguably selective. To investigate the magnitude of the error introduced by not using the proper PLFA signature, we repeated the fitting procedure with the PLFA composition from drinking water DISCUSSION The PLFA composition we determined in algal cultures The PLFA composition of Haptophyta I resembled that of the 2 Dinophyceae; for this reason these 2 groups were combined for the estimation of group abundance. The pigment peridinin is unique to the Dinophyceae. However, Dinophyceae are also known that lack peridinin but contain 19'-hexanoyloxyfucoxanthin and chl c, alloxanthin and chl c, fucoxanthin, chl c 1 and chl c 2 , or chl b Several studies have described variations in fatty acid composition in natural waters and have used fatty acid composition to describe phytoplankton community structure In our analyses, the ratio file As pointed out by The culture conditions were chosen to approximate environmental conditions in the estuary. The cultivation temperature of 15°C was in the middle of the temperature range of the estuary, which was 11, 20 and 14°C in April, July and October respectively. Similarly, the relatively low irradiance of 40 µmol m -2 s -1 was considered appropriate for this very turbid estuary. The fatty acid composition of algae is known to vary in response to changes in environmental parameters such as temperature Especially in a net heterotrophic estuary such as the Scheldt Zooplankton were ignored in our analysis, although some zooplankton were seen in the samples. It was assumed that their abundance was so low that their importance was minor compared to phytoplankton and bacteria. This assumption is supported by the good agreement between pigment and PLFA data. A possibility would have been to remove zooplankton by prefiltering the samples over a zooplankton filter. We chose not to do this since many of the algal species formed large colonies similar in size to the zooplankton or were attached to larger particles and would thus also have been removed by a zooplankton filter. The phytoplankton composition calculated from pigment and PLFA data As the result of tidal forcing and the morphology of the estuary, the turbidity maximum of the Scheldt estuary is around 2 to 8 PSU. This high turbidity results in an unfavorable light climate for phytoplankton. Bacterial growth and relative abundance are usually high in this area It should be remembered that the results in Some degree of uncertainty is inherent in comparisons of cell counts, pigment data and PLFA data, with errors being associated with all methods. Uncertainty in estimations of group abundance using CHEMTAX is commonly assumed to be in the range of 25% for individual groups (e.g Acknowledgements. We thank E. Boschker, J. Peene, P. van Rijswijk, M. Houtekamer, J. Nieuwenhuize, U. Wollenzien, J. J. Middelburg and the crew of the RV 'Luctor' for their help and assistance during the measurements and M. Doeleman for the 18S molecular sequencing. We also thank 3 anonymous reviewers whose remarks helped considerably to improve this manuscript. This work is part of a Flemish-Dutch cooperation for Sea Research (VLANEZO

Prevention of tuberculosis in macaques after intravenous BCG immunization

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette–Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography–computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis