Micronekton were collected using a 10 m2 multiple opening-closing net and environmental sensing system (MOCNESS) at Station ALOHA (22.75˚N, 158˚W) in March and August of 2011 and in February and September of 2014 with a few samples from other locations around Oahu in 2011 (Choy et al 2015). Micronekton were collected over five depth zones between the surface and 1500 m: 0 – 100 m, 100 – 500 m, 500 – 700 m, 700 – 1000 m and 1000 – 1500 m. At sea, micronekton were sorted and identified to the most specific taxonomic level, then measured and photographed. Standard length measurements were taken for fish, carapace length and total length were taken for crustaceans and both mantle length and total length were taken for cephalopods. For most fishes, white muscle tissue was removed and frozen in a cryovial in liquid nitrogen. Small fishes, crustaceans and gelatinous organisms were frozen whole or individuals were pooled for sufficient tissue required for stable isotope analysis. Specimens were transferred to a ‑80˚C freezer until the samples could be prepared for stable isotope analysis.
Eighty-three samples (individual specimens or small groups of conspecifics) were selected for stable isotope analysis. Samples selected for stable isotope analysis represented different combinations of trophic strategies (suspension feeding, zooplanktivores, micronektonivores), depth guilds (epipelagic, mesopelagic, bathypelagic) and migrating behaviors based on available ecological information (e.g., Clarke 1973, Maynard 1982). Each sample was freeze-dried and ground using a ceramic mortar and pestle. For bulk tissue carbon and nitrogen isotope analysis approximately 0.5 mg of each sample was weighed and placed into a tin boat. Carbon and nitrogen isotopic compositions were determined using an isotope ratio mass spectrometer (DeltaPlusXP) coupled to an elemental analyzer (Costech Model 4010). Isotopic ratios are given in δ-notation relative to the international standards VPDB and atmospheric N2. Accuracy and precision were 0.2‰ based on glycine and homogenized fish tissue reference materials analyzed every ten samples. The isotopic compositions of the reference materials have been extensively characterized using NIST certified reference materials in the UH laboratory and verified independently in other isotope laboratories.
Amino acid-specific stable N isotope composition was determined on approximately 15 mg (dry weight) of each sample underwent acid hydrolysis and derivatization yielding trifluoroacetic (TFA) amino acid esters following the methods of Popp et al. (2007) and Hannides et al. (2009b).; The nitrogen isotope composition of the trifluoroacetic amino acid esters were determined using an isotope ratio mass spectrometer (Thermo Scientific Delta V Plus or Thermo Scientific MAT 253 IRMS) interfaced with a Thermo Finnigan GC-C III. Samples were injected onto a BPx5 forte capillary column (60m x 0.32 mm x 1.0 µm film thickness) at an injector temperature of 180˚C with a constant helium flow rate of 1.4 mL/min. The column was initially held at 50˚C for two minutes and then increased at a rate of 15˚C/min to 120˚C. Temperature was then increased at a rate of 4˚C/min to 195˚C, then to 255˚C at a rate of 5˚C/min and finally to 300˚C at a rate of 15˚C/min, holding at the final temperature for eight minutes. Each sample was analyzed in triplicate and co-injected with the reference compounds norleucine (Nor) and aminoadipic acid (AAA) of known isotopic composition. A suite of pure amino acids of known nitrogen isotopic composition (Ala, Thr, Ile, Pro, Glu, and Phe) was also injected every three runs as an extra measure of accuracy for the instrument. Reference compounds Nor and AAA, as well as the suite of amino acids, were used to normalize the measured isotope values. Standard deviation for all amino acids averaged ±0.4‰ (range 0.0-3.1‰).
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