Sampling

Plankton samples were obtained during Leg 8 of the Malaspina-2010 expedition on R/V Sarmiento de Gamboa (January-March 2011), on a transect predominantly along 24ºN, between the Canary Island and Florida. Briefly, plankton samples were collected by vertical tows of a microplankton net (40 µm mesh size) and a mesoplankton net (200 µm mesh size) through the upper 200 m of the water column. Sampling was between 10:00 and 16:00 h GMT. Plankton was separated into five size fractions (40–200, 200–500, 500–1000, 1000–2000 and 2000 µm) by gentle filtration of the samples by a graded series of nylon sieves (2000, 1000, 500, 200 and 40 µm). Large gelatinous organisms were removed before filtration. Aliquots for each size fraction were collected on pre-weighed glass-fiber filters, dried (60ºC, 48 h) and stored in a desiccator before determination of biomass (dry weight), carbon and nitrogen content and natural abundance of stable carbon and nitrogen isotopes ashore. Nominal values of the individual size of organisms in each size fraction were estimated as the geometric mean of the values defining each size interval and expressed as carbon content (µg C) in a logarithmic scale

Bulk δ15N analysis

After determination of dry weight, finely ground aliquots of each size fraction were packed in tin capsules for elemental and stable isotope analysis by conversion into CO2 and N2 in an elemental analyzer (Carlo Erba CHNSO 1108) coupled to an isotope-ratio mass-spectrometer (Finnigan Mat Delta Plus).

Compound-specific amino acid δ15N analysis

Samples for CSI-AA were selected to span gradients in 15Nbulk values. We chose plankton from four sampling stations in each of the three zones (eastern, central and western regions).  Individual samples were then pooled (quantitatively, so that each subsample was represented equally in the final composite) to have enough material in each size fraction for CSI-AA. In total 15 samples in the transect were chosen for CSI-AA. Approximately 1 mg of total dry plankton material was then hydrolyzed for subsequent analysis.

The δ15N values of individual AAs were measured via GC-IRMS, after 6 N HCl acid hydrolysis and the formation of TFA ester derivatives following previously published methods.  Briefly, amino acids were liberated by hydrolysis (6 N HCl, 20 hr at 110uC) under nitrogen, and TFA derivatives subsequently prepared from free AA: isopropyl esters were made with a 1:5 mixture of Acetyl Chloride (AcCl):2-propanol (110uC, 60 minutes), and then acylated using a 1:3 mixture of Dichloromethane:Trifluroacetyl acetate (DCM:TFAA) (100uC, 15 minutes). Derivatized AAs were dissolved in DCM to a final ratio of approximately 2 mg of original dry sample to 250 ml DCM. After derivatization, samples were analyzed by a thermos Trace Ultra gas chromatograph coupled to a Finnegan Delta-Plus isotope ratio mass spectrometer (GC-IRMS). AAs were separated using a 50 m, 0.32 ID Hewlett Packard Ultra-1 column with 1 mm film thickness.  AAs were measured based on n = 4 injections, and the average mean deviations for individual AA d15N measurements across all sample replicates was 0.5%.

Under these conditions, we determined δ15N values for 12 AAs: glutamic acid + glutamine (Glx), aspartic acid + asparagine (Asp), alanine (Ala), Isoleucine (Ile), Leucine (Leu), Proline (Pro), valine (Val), glycine (Gly), serine (Ser), Lysine (Lys), phenylalanine (Phe), and Threonine (Thr). Each AA was run four times on the GC-IRMS..  AA values were categorized and presented in 3 groups, based on their relative 15N values changes with trophic transfer: the source AAs (Gly, Ser, Lys, Phe), the trophic AAs (Glx, Asp, Ala, Ile, Leu, Pro and Val), and one “metabolic” AA (Thr).

Trophic position and ΣV 

To calculate CSI-AA based TP of plankton we used the most widely used current equation and TEF value, based on the isotopic offset between Glx and Phe:

TP  = (δ15NGlx – δ15NPhe – 3.4)/7.6 +1

where δ15NGlx and δ15NPhe are measured values, +3.4‰ is the assumed isotopic difference between the Glx and Phe in primary producers, and +7.6‰ is the assumed 15N enrichment in Glx relative to Phe with each trophic transfer from food source to consumer (TEF value). The standard errors in the estimation of TP, computed by propagation of analytical error in the individual AA determinations, did not exceed 0.1 TP.

The δ15N value of total hydrolysable AAs (δ15NTHAA) is used as a proxy for total protein δ15N value, and was estimated as the molar-weighted average of individual δ15N values:

δ15NTHAA = Σ (δ15NAA * mol% AA)

where δ15NAA is the δ15N value of each individual AA measured and mol%AA is the molar percentage contribution of each AA. In our study we used the δ15N value of each individual AA and mol%AA were obtained from Lehman (2009).

The degradation index ΣV is a measure of the relative resynthesis of the original autotrophic AA pool in detritus or different organisms (plankton size fractions, in our case) was for each size individual fraction sample as the mean deviation of δ15N of individual trophic amino acid, from their average:

ΣV  = Σ (AAi – Avg trp) / n

Where AAi were individual δ15N amino acid values, Avg trp is the average value and n the total number of trophic amino acids.