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1 This article was downloaded by: [CDL Journals Account] On: 23 January 2010 Access details: Access Details: [subscription number ] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Isotopes in Environmental and Health Studies Publication details, including instructions for authors and subscription information: Stable nitrogen isotopes in essential versus non-essential amino acids of different plankton size fractions Natalie Loick a ; Matthias Gehre b ; Maren Voss a a Baltic Sea Research Institute Warnemünde, Rostock, Germany b Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany To cite this Article Loick, Natalie, Gehre, Matthias and Voss, Maren(2007) 'Stable nitrogen isotopes in essential versus nonessential amino acids of different plankton size fractions', Isotopes in Environmental and Health Studies, 43: 4, To link to this Article: DOI: / URL: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Isotopes in Environmental and Health Studies Vol. 43, No. 4, December 2007, Stable nitrogen isotopes in essential versus non-essential amino acids of different plankton size fractions NATALIE LOICK*, MATTHIAS GEHRE and MAREN VOSS Baltic Sea Research Institute Warnemünde, Seestr. 15, Rostock, Germany Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research UFZ, Permoserstr. 15, Leipzig, Germany (Received 07 March 2007; in final form 22 July 2007) The stable nitrogen isotope values (δ 15 N) of the essential amino acid (EAA) leucine and the δ 15 N values of six non-essential amino acids (NEAAs) from plankton size fractions from the South China Sea (SCS) were analysed. Data from the SCS were collected during two cruises in July 2003 and 2004 onboard of RV Nghien Cuu Bien. The δ 15 N values of alanine, aspartic acid, glutamic acid and leucine increased with size at all sites. The δ 15 N of glycine did not increase with size, the δ 15 N of tyrosine increased with size only at offshore stations and the δ 15 N of proline increased with size only at inshore stations. We found highly significant correlations between the δ 15 N ratios of leucine to the δ 15 N ratios of glutamic acid, proline, alanine, tyrosine and aspartic acid at oligotrophic sites of enhanced nitrogen fixation. In contrast thereto these correlations were less distinct or absent at more eutrophic sites of low nitrogen fixation. A comparison with an independent data set from the tropical North Atlantic revealed intriguing similar patterns. We interpret these patterns as result of the connected metabolism of EAA and NEAA in zooplankton at sites of nitrogen limitation. Keywords: Amino acids; Internal nitrogen recycling; Nitrogen-15; Nitrogen fixation; South China Sea; Zooplankton 1. Introduction The recent application of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) on measuring δ 15 N in natural amino acid samples by McClelland and Montoya [1, 2] shows the potential of this technique for direct determination of amino acid dynamics between zooplankton and diet. Their experimental results deepened the understanding on how the food source and trophic-level information in total nitrogen δ 15 N measurements is accomplished. According to their feeding experiments with the algae Tetraselmis suecica and its consumer the rotifer Brachionus plicatilis, bulk δ 15 N values in plankton are determined by Revised version of a paper presented at the 27th Annual Meeting of the German Association for Stable Isotope Research (GASIR), 4 6, October, 2006, Freiberg, Germany. *Corresponding author. Natalie Loick, Aalstecherstr. 3, Rostock, Germany. Tel.: ; Fax: ; nloick@yahoo.com Isotopes in Environmental and Health Studies ISSN print/issn online 2007 Taylor & Francis DOI: /

3 282 Natalie Loick et al. large changes in the δ 15 N values of some amino acids and small changes of others. Glutamic acid, alanine and aspartic acid showed the largest difference in δ 15 N values of , whereas phenylalanine, serine, glycine and tyrosine showed the smallest differences of 0.3 to 0.9 between diet and consumer. These differences in amino acid δ 15 N resulted in moderate differences in bulk δ 15 N between diet and consumer of But this may not be the only information hold in the δ 15 N values of amino acids of zooplankton. Amino acids allocate a key function in the metabolism of tropical marine zooplankton as % of the body dry weight are proteins and the metabolism is protein based [3, 4]. Zooplankton developed different strategies to maintain its metabolism even under unfavourable conditions and copepods are capable to keep constant C:N ratios and amino acid composition even under low food conditions [5 7]. In order to avoid such a misbalance, it has been suggested that the ammonium excretion is reduced [8]. This may be achieved by optimisation of the intracellular amino acid metabolism which means here the use of essential amino acids (EAAs) to synthesise non-essential amino acids (NEAAs) rather than being excreted as ammonium [9]. So far, there has been no method to detect the translocation of amino groups from EAAs to non-neaas. Nitrogen limitation, especially in oligotrophic areas such as the South China Sea (SCS) or the tropical North Atlantic (NA), may cause a shortage in amino acid supply and disturb the optimal amino acid balance in crustaceans, especially as 10 amino acids are essential [10]. We think that the GC-C-IRMS analysis of EAAs and NEAAs holds the potential to detect the translocation of amino groups in natural plankton samples. Here we want to show, also by comparing our data with those from the NA, that δ 15 N amino acid values may indicate that the EAA and NEAA metabolisms are connected under nitrogen limitation. For the first time, we show stable nitrogen isotope data from seven amino acids including the EAA leucine from six different plankton size fractions from the southern SCS. We found strikingly similar patterns in the re-analysis of amino acid δ 15 N data from McClelland and Montoya [2] from two sites in the tropical NA that also differed in N 2 - fixation intensity. We discuss these results against the background of different environmental parameters and explain why they may be the consequence of intensified translocation of amino groups from EAA to NEAA in zooplankton. 2. Methods 2.1 Field sampling During southwest monsoon 2003 and 2004, observations on a fixed station grid with RV Nghien Cuu Bien took place in the Vietnamese upwelling area at 11 to 12.7 N and 109 to E from 10 to 16 July 2003 and from 8 to 26 July 2004 (figure 1). Net plankton was collected in vertical tows with JUDAY-nets (45 and 200 μm mesh, respectively, diameter 0.37 m) through the upper 100 m of the water column. Plankton was separated into size fractions by passing it through 166 μm and 55 μm, 2500 μm, 1500 μm, 1000 μm, 330 μm Nitex sieves. Samples were rinsed plenteously during the separation process to break up and remove any Trichodesmium colonies caught in the sieves. Prior to drying at 60 C and storage in aluminium foil, all size fractions were qualitatively inspected under a microscope. 2.2 Plankton sample selection for GC-C-IRMS analysis Four sets of size-fractioned plankton samples were chosen for stable nitrogen amino acid analysis based on the position of the sampling stations within or outside the upwelling area

4 N isotopes in EAA versus NEAA 283 Figure 1. Stations chosen for amino acid analysis in- and outside the upwelling area as indicated by the dotted line after Dippner et al. [13]. Stations 11, 12, 33a and 33 were sampled during SWM 2003 and stations 21, 56 and 65 during SWM Also included are the sea surface salinities <33.2 indicative for the influence of Mekong waters [17]. (figure 1). Stations within the upwelling area included stations 11 and 12 (2003) and station 21 (2004). We pooled the plankton size fractions from stations 11 and 12 to get enough material for the amino acid analysis (figure 2). Pooling criteria included similarities in bulk δ 15 N values (figure 2), physico-chemical parameters as well as chlorophyll a distributions at the stations (figures 3 and 4). Stations outside the upwelling area included stations 33a and 33 (2003) and stations 56 and 65 (2004). We pooled the plankton size fractions from stations 33a and 33 and added size fraction from station 56 to better cover the size spectrum, Figure 2. δ 15 N bulk values from July 2003 (a) and 2004 (b) from stations inside (black symbols) and outside (open symbols) the upwelling area. Encircled are the samples used or pooled for analysis of δ 15 N in individual amino acids.

5 284 Natalie Loick et al. Figure 3. Vertical salinity, temperature, nitrate and Chl a distributions at stations within (closed symbols) and outside (open symbols) the upwelling area during SWM 2003 (left panels) and SWM 2004 (right panels). Note the different Chl a scales for 2003 and 2004 data.

6 N isotopes in EAA versus NEAA 285 Figure 4. Normalised depths integrated Chl avalues over the upper 70 m of the water column from (a) SWM 2003 and (b) SWM The black dots mark the position of the sampling stations for the amino acid analysis. Contour interval = 0.1mg/m 3. of which especially size fraction μm was poorly covered due to lack of plankton material. All δ 15 N-bulk measurements were made by a continuous-flow isotope mass spectrometer (Finnigan Delta Plus) via a Conflow II open split interface. Calibration for the total carbon and nitrogen isotope determination was done daily with an acetanilide standard. IAEA-N3 (KNO 3 ) was used as the standard for nitrogen. A laboratory internal standard (Peptone, Merck) was run for every sixth sample. The peptone standard indicated an analytical error associated with the isotope measurements of <0.2 for nitrogen-stable isotopes. Two replicates were analysed from each sample. Results have been published elsewhere [11]. Here we will describe only the additional steps for GC-C-IRMS analysis. 2.3 Sample preparation and GC-C-IRMS analysis Analysis of stable nitrogen isotopes in individual amino acids was done for 20 samples as shown in figure 2. Samples were prepared for GC-C-IRMS analysis of amino acids by acid hydrolysis followed by derivatization to trifluor-acetylated-amino acid (TFA) esters [12]. In brief, between 10 and 50 mg of dried sample was placed in a mm glass tube with a polytetrafluoroethylene (PTFE)-lined cap and hydrolysed with ultra pure 6 μm (32 %) hydrochloric acid at 110 C for 24 h. The hydrolysate was filtered through 0.2 μm sterile glassfibre filters and evaporated to dryness with a rotary evaporator. Iso-propanol and acetylchloride (4:1) were added to the dried samples and esterification reaction took place at 110 C for 1 h. After evaporation to dryness under a gentle stream of N 2 gas, 500 μl dichlormethane (DCM, ultrasterile, H 2 O < %) and 500 μl trifluor-aceticacid-anhydride were added (exotherm reaction). Acetylation reaction took place overnight at room temperature (20 C). The remaining liquid was dried under a stream of N 2 gas. Derivatized amino acids were solved in 500 μl ofdcm and stored at 4 C before GC-C-IRMS analysis within the next 7 days. This preparation scheme allows the analysis of 13 common amino acids plus trans-4- (aminomethyl)-cyclohexanecarboxylic acid (internal standard). In the acidic milieu, the δ 15 N values of asparagine and glutamine are only measured in mixture from the acids and the accompanying amides and could not be separated. To simplify matters, we will uniformly term them aspartic acid (asx) and glutamic acid (glx) in the following. Separation problems with the esterification/tfa derivatization method may occur for phenylalanine, lysine and tyrosine.

7 286 Natalie Loick et al. Here, peaks were clearly separated for tyrosine but not for phenylalanine and lysine. The advantage of this derivatization method compared with the esterification/acetylation method is that a relatively simple, rapid reaction set-up is possible for a two-step reaction without additional cleaning of the derivates [12]. The stable isotopic composition of nitrogen in TFA derivatives of amino acids were analysed by GC-C-IRMS (GC: HP series II, Hewlett Packard, USA; C: Combustion Interface; IRMS: MAT252). Mixes of amino acid derivatives from samples were injected into the GC, separated on a BPX5 (SGE) capillary column (50 m 0.32 mm inner diameter, 0.5 μm film thickness; SGE, Australia), combusted, reduced and finally passed through the cold trap to remove water and CO 2 before introduction to the mass spectrometer. Nitrogen isotope ratios for each amino acid in a mix were measured sequentially. Injections were done two to five times per sample splitless or in dilution of 15:1, 10:1, 5:1, 3:1 or 2:1 at 250 C and contained 1 5 μl of sample to optimize peak separation and shape. The GC temperature programme for each run was as follows: initially 50 C, held for 1 min; increased at steps of 10 C/min 1 to 80 C, the temperature was held for 5 min; increased from 80 to 210 Cat3 C/min, held for 5 min; increased from 210 to 280 Cat15 C/min, held at 280 C for 5 min. Standard mixtures of amino acids were run after every 10th sample through the entire analytical procedure to confirm that the sample processing including the hydrolysis and derivatization steps leads to complete derivatization of the amino acids by checking peak areas and δ 15 N values of the internal standard. The external standards then have been used as correction standards. Analytical error associated with isotope measurements of mixtures of purified amino acids through our derivatization were <1 for external standards. As check on our sample processing, we added an internal standard (trans-4-(aminomethyl)- cyclohexanecarboxylic acid) to each sample before the hydrolysis step. Analytical error associated with our derivatization and analytical protocols were <0.3 for these internal standards. All natural samples were injected and analysed two to five times. Satisfactory analysis of natural samples were accomplished for seven amino acids including alanine (ala), glycine (gly), leucine (leu), proline (pro), aspartic acid (asx), glutamic acid (glx) and tyrosine (tyr) with standard deviations <1.5. All isotope abundances are expressed in δ notation as follows: δx( ) =[(R sample /R standard ) 1)] 10 3, where X is 13 Cor 15 NandRisthe 13 C: 12 C or 15 N: 14 N ratio. Stable nitrogen isotope data from different amino acids from this study as well as from the study by McClelland and Montoya [1] were used to relate the δ 15 N of the EAA leucine to the δ 15 N of the NEAA alanine, aspartic acid, glutamic acid, glycine, tyrosine and proline. The classification into EAA and NEAA was carried out after Guillaume [8]. The data from McClelland and Montoya [1] is the only other data set, besides this one, on δ 15 N in amino acids from plankton size fractions. McClelland and Montoya [1] measured the δ 15 N of amino acids in three plankton size fractions ( μm, μm, μm) at two stations (21, 30) from the tropical NA. McClelland and Montoya [1] used N-pivaloyl-i-propyl-amino acid esters that show small differences in δ 15 N values compared with the TFA esters as described by Hofmann et al. [12]. However, here we compare the ratios of their EAA versus NEAA δ 15 N values rather than the absolute δ 15 N values in which case the different derivatization techniques may be disregarded. 3. Results 3.1 Hydrography Stations 33a, 33 and 65 were in the vicinity of Mekong-influenced waters with surface salinities <33.2 (figure 1). The inshore stations 11, 12 and 21 had higher salinities of >33.9 (stations

8 N isotopes in EAA versus NEAA and 12) and >34.2 (station 21) and lower temperatures of <28 C in the upper 100 m compared with salinities >33.0 (stations 33a and 33), >33.5 (station 56), >33. 1 (station 65) and temperatures <29 C at the offshore stations (figure 3). The nitrate concentrations were depleted in the upper 40 m, except for stations 12 and 21, where only the upper 20 m were depleted (figure 3). Below the nutricline nitrate concentrations generally increased to >17 μmol/l below 150 m. Chlorophyll a (Chl a) profiles showed subthermocline maxima at Figure 5. δ 15 N of different amino acids from six plankton size fractions covering a size range from 55 to >2500 μm comprised from both SWM seasons. Black circles ( ) indicate the samples from upwelling influenced stations and open circles (O) from non-upwelling influenced stations. Black lines show the natural logarithm-regression lines.

9 288 Natalie Loick et al m depths. Only at station 12, a surface maximum was found. Although an in offshore trend was less visible from the vertical Chl a profiles, the depth normalised (to maximum 70 m depth) integrated Chl a concentrations showed higher values in the upwelling area with concentrations >0.5mg/m 2 compared with offshore waters, thus reflecting the differences in productivity (figure 4). In July 2003, the maximum value of 0.8 mg Chl a/m 2 was much lower than the maximum value during 2004 of 2.1 mg Chl a/m 2, indicating the weak upwelling intensity during Stations 11 and 12 had integrated Chl a values of 0.5 mg/m 3 compared with slightly lower values around 0.3 mg/m 3 at stations 33a and 33 in July A stronger difference between in- and offshore values was found in July 2004, when station 21 had an integrated Chl a value of 2.1 mg/m 2 compared with <0.4 mg/m 2 at stations 56 and 65. A detailed description of the hydrological situation during SWM has been published by Dippner et al. [13]. 3.2 δ 15 N in plankton Bulk δ 15 N values generally increased logarithmically with plankton size (figure 2). Plankton from upwelling influenced sites was enriched in 15 N compared with plankton from outside the upwelling area during both years. In July 2004, δ 15 N values of the three smallest size fractions (55 166, , μm) from the offshore sites were 2 lower than in July 2003, whereas the δ 15 N values from inside the upwelling area were rather similar between 4.5 and 6. Amino acid concentrations have not been analysed; however, we assume that the amino acid concentrations increased with plankton size as C:N ratios decreased from 7 to 4 from the smallest to the largest size fraction (data not shown). According to literature, all measured amino acids are most abundant in zooplankton compared with other amino acids, except for tyrosine which has been found in lower concentrations in zooplankton [14, 15]. δ 15 N in the amino acids alanine, aspartic acid, glutamic acid and leucine increased logarithmically with plankton size throughout the area (figure 5a d). δ 15 N in tyrosine only increased Figure 6. Correlations of δ 15 N of different amino acid from six plankton size fractions from stations inside (filled symbols) and outside (open symbols) the upwelling area from SWM 2003 and Data points are comprised from six different plankton size fractions from SWM 2003 and Black lines give the linear correlations of the different ratios. For details, see table 1.

10 N isotopes in EAA versus NEAA 289 significantly in the upwelling area, whereas proline increased significantly only outside the upwelling area (figure 5f and g). In contrast to the other amino acids, δ 15 N in glycine did not increase with plankton size (figure 5e). Although most of the seven amino acids increased with size like the bulk plankton δ 15 N, they did not reflect the lower δ 15 N values outside the upwelling area as found in bulk δ 15 N. Only δ 15 N in tyrosine followed this pattern and had δ 15 N values that were 2 6 depleted outside compared with inside the upwelling area (figure 5f). 3.3 δ 15 N leu : δ 15 N NEAA In the SCS data, the δ 15 N of alanine, aspartic acid glutamic acid and proline were highly significantly correlated to the δ 15 N of leucine outside the upwelling area (figure 6, table 1). Table 1. Regression equations (y) for linear correlations between the δ 15 N ratios of six different NEAAs and leucine from upwelling and non-upwelling sites. Amino acid y d.f. (= n 2) r 2 p-value Inside the upwelling area Alanine 1.7x <0.001 Aspartic acid 0.3x <0.05 Glutamic acid 0.01x n.s. Glycine 0.5x n.s. Tyrosine 0.02x n.s. Proline 0.2x n.s. Outside the upwelling area Alanine 1.3x <0.01 Aspartic acid 0.8x <0.01 Glutamic acid 1x <0.01 Glycine 0.3x n.s. Tyrosine 0.3x n.s. Proline 0.7x <0.01 n, number of samples; r 2, correlation coefficient; p, significance level; n.s., not significant. Degrees of freedom (d.f.) were chosen according to Bärlocher [23]. Figure 7. Correlations of δ 15 N of different amino acid from three plankton size fractions ( μm) from station 21 (closed symbols) and station 30 (open symbols) from the tropical North Atlantic from McClelland and Montoya [1]. For details, see table 2.

11 290 Natalie Loick et al. Table 2. Regression equations (y) for linear correlations between the δ 15 N ratios of six different NEAAs and leucine from stations 21 and 30 from the NA from McClelland and Montoya [1]. Amino acid y d.f. (= n 2) r 2 p Station 21 Alanine 0.6x n.s. Aspartic acid 0.2x n.s. Glutamic acid 0.6x n.s. Glycine 0.1x n.s. Tyrosine 0.1x n.s. Proline 1.3x <0.001 Statation 30 Alanine 1.2x <0.05 Aspartic acid 1.4x <0.001 Glutamic acid 1.2x <0.05 Glycine 0.5x n.s. Tyrosine 0.2x <0.05 Proline 1.3x n.s. n, number of samples; r 2, correlation coefficient, p, significance level; n.s., not significant. Degrees of freedom (d.f.) were chosen according to Bärlocher [23]. In the upwelling area, correlations were found only for δ 15 N leu :δ 15 N ala and δ 15 N leu :δ 15 N asp (figure 6). The details of the regression lines are given in table 1. In the tropical NA, the correlations of δ 15 N leu :δ 15 N NEAA are statistically less significant due to only three data points. Nevertheless, the different ratio patterns between data from station 30 compared with station 21 are striking (figure 7, table 2). Whereas at station 30 positive correlations between the ratios of δ 15 N leu to the δ 15 N of alanine, aspartic acid, glutamic acid and tyrosine were found, no such correlations were found at station Discussion 4.1 Nitrogen bioavailability Nitrogen fixation is indicative for nitrogen limitation of primary production and occurs at many marine sites when the water column is depleted in nitrate. N from N fixation may become an important additional N source also for higher trophic levels during nitrogen limitation as shown for the Baltic Sea [16]. The SCS of southern central Vietnam and the tropical NA are sites of intense nitrogen fixation [17, 18]. Zooplankton of Vietnam and in the tropical NA may receive up to 13 and 51 %, respectively, of its nitrogen from nitrogen fixation [11, 17]. The seven sampling stations in the SCS can be classified into two groups according to the distribution of temperature, salinity, depths of the nutricline, Chl a concentration, primary production rates, N 2 -fixation rates and stable nitrogen isotopes in bulk plankton. The coastal near stations 11, 12 and 21 are eutrophic sites of enhanced Chl a concentrations (figure 4) since upwelling regularly fertilizes a km wide strip along the coast off southern central Vietnam during summer monsoon [13]. The stations 33, 33a, 65 and 56 are oligotrophic sites outside this upwelling area and had a lower nutricline of up to 60 m depth (figure 3), low Chl a concentrations (figure 4), decreased primary production rates and increased N 2 -fixation rates [17] compared with inside the upwelling area. Primary production rates along the coast were 19 and 118 mmol C/m 2 /day in 2003 and 2004, respectively, and 20 and 37 mmol C/m 2 /day, respectively, at the offshore sites [17]. Mean N 2 -fixation rates along the coast were 42 and 19 μmol N/m 2 /day in 2003 and 2004, respectively and 84.5 and 115 μmol N/m 2 /day, respectively, at the offshore stations. Outside the upwelling area, bulk δ 15 N values in the plankton size fractions were generally 1 2 lower than in plankton from inside the upwelling area. This

12 N isotopes in EAA versus NEAA 291 pattern has been found during two monsoon seasons, whereas during intermonsoon season when N 2 -fixation rates were low no differences in bulk δ 15 N in the plankton size fractions have been found in and outside the upwelling area as shown by Loick et al. [11]. The low δ 15 N values in the plankton size fractions have been interpreted as incorporation of N from N fixation and its transfer into higher trophic levels through the food web in consensus with findings from the oligotrophic NA [18]. The δ 15 N values increased with plankton size at any site, although the increases in the consecutive size fractions were smaller than the reported increase generally attributed between consumer and diet [19]. This may have been due to the insufficient resolution of trophic levels and the overlap within one size fraction, species composition, food quality and quantity, N turnover or amino acid composition [11]. Despite these factors, only trophic fractionation can cause the general increase in δ 15 N with size and is confirmed by other studies [19, 20]. On the basis of these δ 15 N patterns in the plankton size fractions, we conclude that the nitrogen supply of the plankton community has been less favourable at the offshore sites than inside the upwelling area during both seasons. The offshore sites therefore may comply with the environmental perquisites to anticipate an enhanced coupling between the synthesis of NEAAs from EAAs in its plankton community. In the following section, these suggestions are discussed in the framework of δ 15 N in amino acids. 4.2 Amino acid δ 15 N pattern We found increasing δ 15 N values of the amino acids alanine, aspartic acid, glutamic acid and leucine with size at in and offshore sites (figures 5a d). This is similar to the increase in δ 15 N of the total nitrogen values and can be explained by trophic enrichment [19]. An increase in δ 15 N values of organic matter has been attributed to fractionation during transamination and desamination of amino acids [21, 22] and lab experiments showed that increases in 15 N appear to be the greatest in amino acids that are extensively involved in nitrogen transfer [1]. No increase with size was found for glycine (figure 5e). The values for tyrosine increased significantly only at the inshore sites and for proline only at the offshore site (figures 5f and g). Besides trophic enrichment, differences in the δ 15 N of amino acids have also been related to the δ 15 N of the principal nitrogen source. Glycine and tyrosine were found to remain largely unaltered by trophic enrichment and may reflect the δ 15 N of nitrogen sources such as nitrate or N 2 [1]. The lower δ 15 N values in tyrosine at the offshore stations in the SCS therefore may reflect the additional incorporation of N 2 (δ 15 N of 0 ) at these stations compared with the principal use of nitrate (δ 15 Nof3.6 ) along the coast. This is in consensus with findings by Voss et al. [17] and Loick et al. [11]. The amino acid δ 15 N patterns from the SCS are very similar to those in plankton size fractions from stations 21 and 30 in the tropical NA by McClelland and Montoya [1]. The two stations in the tropical NA differed in the abundance of the cyanobacterium Trichodesmium spp. and the δ 15 N of total nitrogen of different plankton size fractions. At the tropical NA station 30, high abundances of Trichodesmium of trichomes m 2 were found compared with < trichomes m 2 at station 21 [2]. The isotope values of the total nitrogen from three plankton size fractions were generally 1.5 depleted at station 30 compared with station 21. McClelland et al. [2] interpreted this as an incorporation of nitrogen from nitrogen fixation into higher trophic levels at station 30 opposed to station 21 where nitrate seemed to have been the principal nitrogen source. We take their findings as an indication for more severe N limitation at station 30 compared with station 21. A coupling of the metabolism of NEAAs to the metabolism of EAAs therefore may have been enhanced in plankton from station 30 compared with station 21.

13 292 Natalie Loick et al. 4.3 Ratios of δ 15 N leu vs δ 15 N NEAA Despite the large environmental differences between the inshore and offshore sites in the SCS and stations 30 and 21 from the tropical NA, respectively, we found no differences in the δ 15 N distribution of amino acids in association with the plankton size. However, we found consistent differences in the correlation of the δ 15 N of the EAA leucine to the δ 15 N of the NEAAs from 20 samples from seven stations at sites of high and low nitrogen fixation in the SCS. The comparison with the independent data set from the tropical NA revealed intriguing similar patterns (figures 6 and 7). For the first time, it is shown that the δ 15 N of leucine is positively correlated to the NEAAs alanine, aspartic acid, glutamic acid, proline and tyrosine at sites of enhanced nitrogen fixation (tables 1 and 2). No such correlations were found at sites of sufficient nitrogen supply for the planktonic food web. These differences cannot be explained with trophic enrichment or nitrogen source effects but (possibly) with a coupled amino acid metabolism between EAA and NEAAs. The metabolisms of the NEAAs are closely related to each other with glutamic acid in addition to 2-oxoglutarate, pyruvate and oxalacetate as central compounds for synthesis of proline, alanine and aspartic acid [9]. EAAs are excluded from recycling processes and have to be supplied with the food. Once incorporated, they can provide their amino group for the synthesis of NEAA. This mechanism leads to the observed correlation between the δ 15 N of leucine to the δ 15 N of NEAAs. However, a detailed explanation of individual results is beyond the scope of the present study, as the mechanisms are likely complex and largely unknown for zooplankton. 5. Comments and recommendations This is the first time that the δ 15 N values of an EAA have been related to the δ 15 N values of NEAAs. We found different correlation patterns of these ratios between sites of enhanced nitrogen limitation compared with sites of more favourable nitrogen supply from two tropical oceans.we hypothesise that the correlations between the δ 15 N leu and the δ 15 N NEAA are the consequence of a coupled amino acid metabolism between EAAs and NEAAs at sites of nitrogen limitation. In a next step, controlled laboratory experiments have to test this hypothesis. Acknowledgements We acknowledge the support of the captain and officers of RV Nghien Cuu Bien and the excellent cooperation with the working group of Matthias Kästner (UFZ). We thank Katrin Schmidt for encouraging discussions on the data. This work was funded by the Deutsche Forschungsgemeinschaft (DFG/Bo-768). References [1] J.W. McClelland, J.P. Montoya. Trophic relationships and the nitrogen isotopic composition of amino acids in plankton. Ecology, 83(8), (2002). [2] J.W. McClelland, C.M. Holl, J.P. Montoya. Relating low δ 15 N values of zooplankton to N 2 -fixation in the tropical North Atlantic: insights provided by stable isotope ratios of amino acids. Deep-Sea Res.I, 50, (2003). [3] U. Båmstedt. Chemical composition and energy content. In The Biological Chemistry of Marine Copepods, E.D.S. Corner and S.C.M. O Hara (Eds), pp Clarendon, Oxford (1986). [4] P. Mayzaud, R.J. Conover. O: N atomic ratio as a tool to describe zooplankton metabolism. Mar. Ecol. Progr. Ser., 45(3), (1988). [5] C. Guisande, I. Maneiro, I. Riveiro. Homeostasis in the essential amino acid composition of the marine copepod Euterpina acutifrons. Limnol. Oceanogr., 44(3), (1999).

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