Bulk and amino acid nitrogen specific isotope data from particulate organic matter and mesozooplankton (1000-2000 µm) from the Mekong River plume and southern South China Sea

The mean trophic position (TP) of mesozooplankton largely determines how much mass and energy is available for higher trophic levels like fish.  Unfortunately, the ratio of herbivores to carnivores in mesozooplankton is difficult to identify in field samples.  Here we investigated changes in the mean TP of mesozooplankton in a highly dynamic environment encompassing four distinct habitats in the southern South China Sea: the Mekong River plume, coastal upwelling region, shelf waters, and offshore oceanic waters.  We used a set of parameters derived from bulk and amino acid nitrogen stable isotopes from particulate organic matter (POM) and four mesozooplankton size fractions to identify changes in the nitrogen source and structure of the planktonic food web across these habitats.  We found clear indications of a shift in N sources for biological production from nitrate in near-coastal waters towards an increase in diazotroph-N inputs in oceanic waters where diazotrophs shaped the phytoplankton community.  The shift in N source was accompanied by a lengthening of the food chain (increase in the TP), which may provide further support for the connection between diazotrophy and the indirect routing of N through the marine food web.  Our combined bulk and amino acid δ15N approach also allowed us to estimate the trophic enrichment (TE) of mesozooplankton across the entire regional ecosystem.  When put in the context of literature values, our high TE of 5.1‰ suggested a link between ecosystem heterogeneity and the less efficient transfer of mass and energy across trophic levels.

Methods
Samples of seawater and plankton were collected on two legs on R/V Falkor cruise FK160603 (3 June – 18 June, 2016) to the SCS off southern central Viet Nam.

Water samples were obtained using a Seabird CTD-rosette system outfitted with a sensor for chlorophyll a fluorescence (in the following Chl. a).

Samples of particulate organic matter (POM) for elemental and bulk isotope analyses were collected from the same depths as the nutrient samples.  A volume of 1–19 L of seawater was filtered through pre-combusted (450°C for 2 h) 47-mm GF/F filters (0.7 µm nominal pore size) using gentle pressure filtration (5-10 psi).  Filter samples were dried at 60°C onboard and stored over desiccant for analysis ashore.  At selected stations, additional samples of POM for CSIA were collected by gentle pressure filtration of 20-60 L of seawater from either the surface or the chlorophyll-maximum through a series of pre-weighted 47-mm polycarbonate filters (Whatman Nuclepore, 0.2 µm).  Samples were frozen at -20°C and dried at 60°C ashore.

Zooplankton for elemental, bulk isotope, and compound specific isotope analyses were collected in vertical tows around local noon and local midnight with a 0.5 m2 ring net (200 µm mesh size) equipped with a USBL transponder.  We sampled through the upper 100 m of the water column or to ~10 m above bottom at shallower stations.  Animals were size-fractionated using a graded series of Nitex sieves with mesh sizes of 2000 µm, 1000 µm, 500 µm and 250 µm, and dried onboard at 60°C for 48 h.  Dry samples were ground to a fine powder, then stored in pre-combusted aluminum foil (450°C for 2 h) at -20°C for the duration of the cruise.

All elemental concentration and bulk stable isotope measurements of POM and size-fractionated zooplankton samples were made by elemental analysis with coupled isotope ratio mass spectrometry (EA-IRMS).  Analyses were carried out using a Thermo Scientific Delta V Advantage interfaced to a Flash 2000 elemental analyzer in Warnemünde, Germany or a Micromass Isoprime IRMS coupled to a Carlo Erba NA2500 elemental analyzer in Atlanta, GA, USA.  The overall precision of our bulk isotopic analysis is better than ±0.2‰ for δ13C and δ15N for both instruments, which were cross-calibrated by running splits of the same standard sets.

The δ15N values of triflouroacetyl/isopropyl ester (TFA) AA derivatives (Hofmann et al., 2003; Veuger et al., 2005) were measured on a Thermo GC Isolink CN with Trace 1310 (GC), coupled via a ConFlo IV combustion interface (C) to a Mat 253 (IRMS, all Thermo Fisher Scientific GmbH, Dreieich, Germany) in Warnemünde.  The precision of our GC-C-IRMS measurements varied among individual AAs and sample type but the standard deviation of 3-5 analyses typically was ≤1.0‰.  Due to the resource intensive nature of this approach, CSIA analyses were performed on only a subset of POM and zooplankton samples, which included POM from the surface and Chl. a maxima and mesozooplankton from the 1000-2000 µm size fraction.  This size fraction was chosen because it provided good coverage across the various habitats (zooplankton in the larger size fractions were not always present at adequate abundances) and because samples from this size fraction had minimal phytoplankton contamination (explained in more detail below).  In total, 9 POM samples from 6 stations (Supporting Table S1) and 21 zooplankton samples from 15 stations (Supporting Table S2, Fig. 1) were analyzed for CSIA.  Individual nitrogen stable isotopes of 12 AAs included the so-called “source” AAs glycine (Gly), lysine (Lys), phenylalanine (Phe) and tyrosine (Tyr); the “trophic” AAs alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), isoleucine (Ile), leucine (Leu), proline (Pro), and valine (Val); and the “metabolic” AA threonine (Thr), categorized by Germain et al. (2013), McClelland and Montoya (2002), and Chikaraishi et al. (2009) according to the sensitivity of each AA to trophic enrichment in 15N.  Asp and Glu also include the amide forms asparagine and glutamine, respectively, with the N isotopic signature coming only from the α-amino-N from both compounds as the amide N is lost during hydrolysis.  Details of the EA-IRMS and GC-C-IRMS analyses are provided in the Supporting Information S1 of Weber et al.