2.1 Hydrographic data and plankton tow samples
Hydrographic data and plankton tow samples were collected from 0 to 200 m in water depth during the R/V Tansei-Maru cruise (KT-11-25) in the afternoon on October 14, 2011 (full moon phase), at the mouth of Suruga Bay, Japan (34°38.989′N, 138˚33.045'E), where the Kuroshio Current (warm current) flowed off south of Kouzushima Island to the northeast (Fig. 1). Hydrographic data including temperature, salinity, density, dissolved oxygen, and chlorophyll a (Chl a) were measured near the plankton tow sampling site by a conductivity temperature depth sensor (CTD; SBE 9, Sea-Bird Scientific) and a fluorometer (Aquatracka MkIII, Chelsea Technologies Group Ltd.). The upper 200 m was sampled twice using a Vertical Multiple Plankton Sampler (VMPS, Tsurumi-Seiki Co. Ltd.). The tow sampler has a 0.25-m2 opening and 100-μm mesh size (NXX13). Four depth intervals (0–20, 20–50, 50–100, and 100–200 m) were sampled during each tow. The samples were fixed with seawater-buffered 5% formalin in a 50-mL vial. Formalin fixation does not affect δ15N values of amino acids derived from an aquatic consumer (Ogawa et al. 2013).
Samples were poured into petri dishes. Planktonic foraminifers alive at the time of sampling, which were identified by the presence of cytoplasm, were picked from a wet solution by pipetting using a Pasteur pipette, mounted on a microfossil slide, identified, and counted to the species level. Microphotographs of five common species (Neogloboquadrina dutertrei, Pulleniatina obliquiloculata, Globigerina bulloides d'Orbigny, 1826, Trilobatus sacculifer (Brady, 1877), Globigerinoides ruber white (d'Orbigny, 1839)) were taken using a digital camera attached to a binocular dissecting microscope. The maximum diameter was measured using Image J (NIH) to estimate the size-frequency distribution of each species at each depth interval (Additional file 1: Fig. S1). Since other zooplankton were more abundant than planktonic foraminifers, the remaining samples were divided into 2–5 mL subsamples by pipetting from stirred and homogenized samples according to methods described in Omori and Ikeda (1976). Zooplankton alive at the time of sampling, which were identified by the presence of organic matter, were picked from a wet solution by pipetting using a Pasteur pipette and stored in a glass vial filled with seawater-buffered 5% formalin. The zooplankton were identified and counted (Suidosha, Co., Ltd.) and finally classified to the order level. Counts of planktonic foraminifers and zooplankton were converted to the standing stock (SS; the number of individuals m−3 seawater) using the following equations:
$${\text{SS}}_{{\text{planktonic foraminifers}}} = n_{{{\text{sample}}}} \div v_{{{\text{seawater}}}}$$
(1)
$${\text{SS}}_{{{\text{zooplankton}}}} = n_{{{\text{subsample}}}} \div v_{{{\text{subsample}}}} \times v_{{{\text{sample}}}} \div v_{{{\text{seawater}}}}$$
(2)
where n is the count in a sample or subsample (individuals), vsubsample is the volume of a subsample (2–5 mL), vsample is the volume of a sample (50 mL), and vseawater is the volume of seawater. Since the volume of seawater filtered by a plankton net was not obtained using a flow meter, vseawater is calculated by the opening area of a plankton sampler (0.25 m2) multiplied by the water depth interval (20–100 m).
In order to estimate the abundance of particulate organic matter (POM), the remaining residue excluding planktonic foraminifers and zooplankton (referred to as residue POM) and another 5-mL subsample (referred to as total POM) were filtered, wet weighed, and converted to the wet weight of POM (mg m−3) using the following equation:
$${\text{POM}} = \, w_{{{\text{subsample}}}} \div v_{{{\text{subsample}}}} \times v_{{{\text{sample}}}} \div v_{{{\text{seawater}}}}$$
(3)
where wsubsample is the mass of POM in a subsample.
In order to correlate the standing stock data with environmental variables, the median of environmental variables such as temperature, salinity, density, dissolved oxygen, and Chl a, was calculated for each depth interval. Standing stock data of planktonic foraminifers (N. dutertrei, P. obliquiloculata) and zooplankton (Calanoida, Cyclopoida, Poecilostomatoida, Harpacticoida, all copepods, other zooplankton, all zooplankton) and the abundance of POM (residue and total) were square-root transformed. Transformed standing stock data of planktonic foraminifers were correlated with other variables (environmental, zooplankton, POM) using Pearson’s correlations. In addition, the standing stock data were analyzed with a linear model using Chl a and total POM as explanatory variables to examine the effects of biological variables. These statistical analyses were conducted in R 4.1.1 (R Core Team 2021). We also calculated correlations between the standing stock data and the mean of environmental variables. The correlation results using the mean were similar to those using the median.
2.2 Amino acid nitrogen isotope analysis
In this study, we measured δ15N values of the bulk cells (i.e., the sum of the cell cytoplasm, organic membranes, and intracrystalline protein in a shell) of fixed specimens of N. dutertrei and P. obliquiloculata. Since the amount of intracrystalline proteins is fewer than the cell cytoplasm, δ15N values in the bulk cells would be expected to indicate the short-term value of their metabolism. Due to the low number of specimens, fixed specimens from two replicate samples for each water depth interval were pooled for the bulk cell analysis (i.e., one pooled sample of each water depth interval was measured for each species). The number of N. dutertrei and P. obliquiloculata specimens measured for each water depth interval ranged from 18 to 353 and 14 to 137, respectively. Measured test size ranged from 150 to 650 μm for all depth intervals of both species.
The δ15N value of amino acids was determined according to Chikaraishi et al. (2009). Briefly, each specimen was hydrolyzed in 12 M HCl at 110 °C, and then, the hydrolyzate was washed with n-hexane/dichloromethane (3:2, v/v) to remove any hydrophobic constituents. After derivatization with thionyl chloride/2-propanol (1:4, v/v) and subsequently with pivaloyl chloride/dichloromethane (1:4, v/v), the derivatives of the amino acids were extracted with n-hexane/dichloromethane (3:2, v/v). The δ15N value of individual amino acids was determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) using a DeltaplusXP isotope ratio mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled with a 6890 N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) via combustion and reduction furnaces. δ15N value is defined as δ15N = ([15N/14N]sample/[15N/14N]Air − 1) × 1000 and expressed in conventional (‰) against that of air (Air).
The trophic position (TPGlu/Phe) of the sample was calculated based on the following equation proposed by Chikaraishi et al. (2009):
$${\text{TP}}_{{{\text{Glu}}/{\text{Phe}}}} = \, \left( {\left( {\delta^{{{15}}} {\text{N}}_{{{\text{Glu}}}} - \, \delta^{{{15}}} {\text{N}}_{{{\text{Phe}}}} - { 3}.{4}} \right)/{7}.{6}} \right) \, + { 1}$$
(4)
where δ15NGlu and δ15NPhe represent the δ15N values of glutamic acid and phenylalanine, respectively, 3.4 is the isotopic difference between glutamic acid and phenylalanine in primary producers, and 7.6 is the offset of the trophic discrimination factor of these two amino acids per trophic position increase. Although this equation may need modification (particularly for the trophic discrimination factor) for some specific organisms, such modification is not required for organisms in the lower-trophic-level hierarchy of food webs (McMahon and McCarthy 2016). The trophic position is expected to be 1.0 for a “pure” primary producer and 2.0 for a “pure” primary consumer.
Propagation error (potential uncertainty) in TPGlu∕Phe value has been calculated by taking into account the propagation of 1σ for δ15NGlu, δ15NPhe, the isotopic difference between glutamic acid and phenylalanine in primary producers, and the offset of the trophic discrimination factor (TDF) of these two amino acids per trophic position increase in Eq. (4) (Chikaraishi et al. 2009, 2014), and is expressed in the following equation (Kruse et al. 2015):
$${1}\sigma_{{{\text{TL}}}} = \, \left[ {\left( {y^{{2}} {/7}.{6}x} \right) \, + \, \left( {{1}\sigma_{{{\text{TDF}}}} {\text{/TDF}}} \right)^{{2}} } \right]^{{{1}/{2}}}$$
(5)
where x = δ15NGlu − δ15NPhe − 3.4, y = [2(1σm)2 + (1σβ)2]1/2, 1σm = 0.5‰, 1σβ = 0.9‰, and 1σTDF = 1.2‰, after Chikaraishi et al. (2009). Previous studies have indicated that the potential uncertainty in the TPGlu/Phe value calculated via propagation of error is 0.2–0.4 for each trophic level based on an assumed standard deviation of 0.5‰ (1σ) for the observed δ15N values of glutamic acid and phenylalanine (Chikaraishi et al. 2009).