Deep–Sea Research I 197 (2023) 104045

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Deep-Sea Research Part I
journal homepage: www.elsevier.com/locate/dsri

Vertical, spatial, size, and taxonomic variations in stable isotopes (δ13C andδ15N) of zooplankton and other pelagic organisms in the western
North PacificDongwoo Kim a, *, Kanako Amei a, 1, Sota Komeda a, 2, Koki Tokuhiro a, 3, Maki Noguchi Aita b,
Fujio Hyodo c, Atsushi Yamaguchi aaGraduate School of Fisheries Sciences, Hokkaido University, 3–1–1 Minato-cho, Hakodate, Hokkaido, 041–8611, Japan
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2–15, Natsushima-cho, Yokosuka, Kanagawa, 237–0061, Japanc
Research Core for Interdisciplinary Sciences, Okayama University, 3–1–1, Tsushimanaka, Okayama, 700–8530, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
GillnetInterzonal copepods
Latitudinal variation
Subarctic
Transitional domain
Vertical distribution

Stable isotopes (δ13C and δ15N) of zooplankton and other marine organisms are useful for evaluating trophic
levels and the food web structure of marine ecosystems. Further, taxonomic variations and regressions between
δ15N and δ13C can provide details of regional differences in the food web structure. Despite their ecologicalimportance, information on the taxonomic differences in the stable isotopes of various pelagic biota and vertical,
latitudinal, and size variations in the stable isotopes of the mesozooplankton are scarce in the western North
Pacific. In this study, we compared regional differences in the taxonomic variations of the stable isotopes of theall pelagic organisms collected by gillnets and various plankton nets in the subarctic and transitional domains(37–44◦ N) of the western North Pacific during spring. Additionally, we evaluated the vertical, size, and lat­
itudinal variations in the stable isotopes of mesozooplankton in the western North Pacific. Regressions between
δ15N and δ13C were highly significant for the subarctic and transitional domains, while they varied significantly
between the two regions (P < 0.0001, ANCOVA). These regional differences in the stable isotopes were char­acterised by the high δ15N and low δ13C values in the subarctic region, whereas the opposites trend was observedin the transitional domains. Vertical observations from the sea surface to a depth of 3000 m at five stations (29◦ ,31◦ , 33◦ , 37◦ , and 41◦ N) showed that there were no significant changes in the δ13C stable isotope of meso­
zooplankton with depth across the stations. However, for δ15N, the significance of regressions increased withincreasing depths at the subtropical stations (29◦ , 31◦ , and 33◦ N), while no significant trends with depth wereobserved for the subarctic and transitional domain stations (37◦ , 41◦ N). The dominance of interzonal copepods(e.g., Neocalanus spp.) in the mesozooplankton biomass in the subarctic and transitional region may mask thevertical changes in δ15N values in that region. Further, δ13C varied neither with size nor latitude. In contrast,
δ15N showed clear size and latitudinal patterns. Thus, δ15N values increased with increasing zooplankton size
(112 μm < 407 μm < 925 μm), and significant regressions increased with increasing latitude for all three sizeclasses (P < 0.001). Subsequent ANCOVA revealed that the regression did not vary with organism size. Thus, thechanging latitudinal patterns in δ15N values (higher for high latitude) had much greater effects than the size
differences for mesozooplankton.

1. Introduction
In marine ecosystems, zooplankton play two main ecological roles:

1) first, they serve as a mediator of the organic materials and energy
from primary producers to the higher trophic level organisms (fisheries
viewpoint) and 2) second, as an accelerator of the biological pump(oceanographic viewpoint); they are responsible for vertical material* Corresponding author.
E-mail address: dongwoo.kim.s2@elms.hokudai.ac.jp (D. Kim).
1
Present address: Atmosphere and Ocean Research Institute, The University of Tokyo, 5− 1− 5, Kashiwa, Chiba, 277− 8564, Japan.
2
Present address: Takehara Station, Hiroshima University, 5–8–1 Minato-machi, Takehara, Hiroshima, 725–0024, Japan.
3
Present address: Demonstration Laboratory, Marine Ecology Research Institute, 4–7–17 Arahama, Kashiwazaki, Niigata 945–0017, Japan
https://doi.org/10.1016/j.dsr.2023.104045
Received 19 January 2023; Received in revised form 28 March 2023; Accepted 10 April 2023
Available online 11 April 2023
0967-0637/© 2023 Elsevier Ltd. All rights reserved.

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higher trophic level organisms such as fishes and squids in the same
region and period remain scarce. Regarding vertical changes, the study
regions were limited, and little comparable information is available on
the vertical changes in the stable isotope ratio in the subarctic, transi­
tional, and subtropical regions. Regarding latitudinal changes, few at­
tempts have been made based on north–south transects (Richoux andFroneman, 2009; Williams et al., 2014; Horii et al., 2018) and moredetailed studies are needed to accurately evaluate latitudinal changes inthe stable isotopes of zooplankton. Since water masses vary with region,
evaluating their effects on stable isotope ratios is also important.
Historically, for the subarctic and transitional domain of the western
North Pacific, several studies have been conducted on the whole biota,
from zooplankton to fish and squids, by applying both plankton nets andgillnets in latitudinal transect (155◦ E, 170◦ E, and 175◦ E longitudes)(Pearcy et al., 1996; Takagi et al., 1997; Yamaguchi et al., 2005a).
Regarding vertical changes in zooplankton communities, some studies
have analysed vertically-stratified net zooplankton samples from the sea
surface to great depths (maximum 5800 m) at four stations (44◦ N, 39◦ N,
30◦ N, and 25◦ N latitudes in the western North Pacific) to determine
community structure from bacteria to mesozooplankton (Yamaguchi
et al., 2002a, 2004), and access vertical changes in community structure
for specific taxa such as copepods and chaetognaths (Yamaguchi et al.,
2002b; Ozawa et al., 2007). Vertical changes in biomass and chemicalcomposition (C/N ratio) from the surface to the deep sea were also
evaluated based on the same zooplankton samples (Yamaguchi et al.,2005b).
For the latitudinal variations in zooplankton communities, studies
compared changes in community structure along north–south transects
covering the subarctic, transitional, and subtropical regions (Saito et al.,2011; Yamaguchi et al., 2017). Latitudinal variations in zooplankton
community is also characterised by many studies investigating
zooplankton size composition evaluated by the optical plankton counter(OPC), along these transects (Yokoi et al., 2008; Matsuno and Yama­guchi, 2010; Shiota et al., 2013; Mishima et al., 2019). Although thesestudies have presented important insights, they have not analysed stable
isotope ratios, and our knowledge on this topic in the western North
Pacific remains limited.
To this end, the currents study aimed to investigated the following
three topics: (1) evaluate the Δδ15N/δ13C of the pelagic organisms fromzooplankton to fishes and squids, collected by both plankton nets andgillnets in the subarctic and the transitional domain of the western North
Pacific; (2) evaluate vertical changes in δ15N and δ13C from the sea
surface to a depth of 3000 m at fixed stations located between
29◦ 00′ N–42◦ 40′ N, and (3) evaluate latitudinal and size changes basedon the size-fractionated (925 μm, 407 μm, 112 μm) zooplankton samples
collected from the epipelagic zones of stations along the north–southtransect, covering the subarctic to subtropical regions. The results werecompared with those of studies on stable isotope ratios from various
oceans, and the general patterns of stable isotope ratios of pelagic biota
have been discussed. In particular, Δδ15N/Δδ13C, a comparison wasmade with findings from previous studies conducted in this region(western North Pacific) (Aita et al., 2011; Kobari et al., 2022). The
factors causing spatial variations in stable isotope ratios (vertical andlatitudinal changes) are also discussed from the perspective of
zooplankton communities in the western North Pacific.

Abbreviations
ANCOVA analysis of covariance
ANOVA analysis of variance
POM
particulate organic material
SVM
seasonal vertical migration
VMPS
Vertical Multiple Plankton Sampler
Abbreviations
particulate organic matter (POM)
optical plankton counter (OPC)
analysis of covariance (ANCOVA)
North Pacific Standard net (NORPAC net)
Vertical Multiple Plankton Sampler (VMPS)
Conductivity Temperature Depth profiler (CTD)
Temperature vs. Salinity (T-S)

transport from the sea surface to the deep sea via egestion of faecal
particles or vertical migration (Miller and Wheeler, 2012).
Stable isotope ratios (δ15N and δ13C) are useful indices for evaluating
food web structure in marine ecosystems (Aita et al., 2011). It is wellknown that δ15N increases by approximately 3.4‰ each trophic level.
Thus, it is a useful indicator for the trophic level of each taxon within amarine ecosystem (Minagawa and Wada, 1984). On the contrary, δ13C isan indicator of the differences in carbon sources at the origin of primary
production (Hobson et al., 2002). It has been reported that there is acommon regression slope between δ15N and δ13C in terrestrial andmarine environments: δ15N = (1.6 ± 0.2) × δ13C + intercept (Aita et al.,2011). The constant values of Δδ15N/Δδ13C for these various taxa (1.6
± 0.2) are thought to be a reflection of deamino- and decarbonizing
metabolic systems that are common throughout terrestrial and marine
environments (Wada and Noguchi, 2017). While it is interesting, testing
and confirmation of this hypothesis are needed. Spatial changes in thestable isotope ratios of zooplankton present, two dimensions: verticaland horizontal. For vertical changes, δ15N has been reported to increase
with increasing depth in various oceanic regions (Saino and Hattori,
1980; Koppelmann et al., 2003, 2009; Williams et al., 2014; Bode et al.,2015). This is because the frequency of repacking particulate organic
matter (POM) increases with depth (Williams et al., 2014; Richards
et al., 2020). For horizontal, δ15N has been reported to be low at low
latitudes in various oceanic regions (Koppelmann et al., 2003, 2009;
Williams et al., 2014). This is thought to be due to a lower δ15N baselinein the marine food web because the phytoplankton community at lower
latitudes is dominated by cyanobacteria (Trichodesmium spp.) which
possess nitrogen-fixation capabilities (Koppelmann et al., 2003, 2009;
Williams et al., 2014).
A study on the stable isotope ratio of zooplankton in the subarctic
Pacific and its neighbouring waters, was conducted to evaluate Δδ15N/
Δδ13C for the various zooplankton taxa (e.g. copepods, euphausiids,
amphipods, chaetognaths) mentioned above (Aita et al., 2011). As
large-sized interzonal copepods (e.g., Neocalanus spp.) dominate thesubarctic Pacific, various studies have focused on them: east-west,
geographical changes, and interannual variations (Chiba et al., 2012),
regional and interannual variations in the Gulf of Alaska (Kline, 2009,2010), and interannual variations in the British Columbia fjord (El-Sa­
baawi et al., 2009). Regarding vertical changes, studies have investi­
gated the effects of hydrothermal plumes on the Pacific coast of Canada(Burd et al., 2002) and also revealed the vertical changes with depth at
one station each in the subarctic and subtropical western North Pacific(Kobari et al., 2022). Regarding latitudinal variations, Pomerleau et al.
(2014) investigated the epipelagic zooplankton from the Gulf of Alaska
to the western Arctic Ocean. While these studies have revealed impor­tant findings, studies on the stable isotope ratios from zooplankton to the

2. Materials and methods
2.1. Field sampling
During cruises of the T.S. Oshoro-Maru, zooplankton, fishes, andsquids were collected using several sampling methods at depths of
0–150 m at six stations along the north–south transect (located37◦ 15′ N–44◦ 00′ N) along 155◦ E longitude in the western North Pacific
Ocean from 14 to 20 May 2016 (Fig. 1). Zooplankton was collected byvertically stratified sampling using VMPS (mouth opening areas and2

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Deep-Sea Research Part I 197 (2023) 104045

Fig. 1. Sampling stations where the pelagic organ­
isms used in this study were collected, and those of
the other comparable studies (Aita et al., 2011;
Kobari et al., 2022). For the Oshoro-Maru cruise, wetargeted zooplankton and nekton (pelagic fishes andsquids) by using plankton net and gillnet, respec­tively, at the latitudinal transect along 155◦ E during
May 2016 (gray triangles). For the Hakuho-Marucruise, vertical changes in mesozooplankton were
evaluated down to 3000 m by using VMPS, and sizeand latitudinal changes in mesozooplankton wereconducted by NORPAC net from 0 to 200 m along anorth–south transect in the western North Pacificduring October 2017 (solid and with circled symbols).
Dashed lines indicate approximate positions of dif­ferences in water masses (subarctic, transitional
domain, and subtropical region) evaluated by T-S
diagrams in this study (cf. Fig. 2). For the reference
studies, the solid line and solid square indicate thesampling locations of the Oyashio and Warm Core
Ring of Aita et al. (2011), respectively. Solid and open
stars represent the subarctic St. K2 and subtropical St.
S1 of Kobari et al. (2022). The results of these twocomparable studies are shown in Fig. 7.

mesh sizes were 0.25 m2 and 63 μm) and, vertical tows of a NORPAC net(45 cm mouth diameter, 335 μm mesh size) from 0 to 150 m, and hor­izontal towing with a Bongo net (60 cm mouth diameter, 500 μm meshsize) at depth of 5 m. Fishes and squids were collected by horizontal
towing with a larva net (1.3 m mouth diameter, 500 μm mesh size) fromthe sea surface at speed of two knots for 10 min, fishing and a 2500-mlong gillnet deployed overnight. The collected specimens were identified
as species on board, and the whole body of the small species or muscle
parts of the large species were cut out, placed in a plastic bag or vial, andfrozen, and stored at − 20 ◦ C.
During the cruise of R.V. Hakuho-Maru, vertically stratified samplingby VMPS equipped with a 63 μm mesh was made from 12 layers down to3000 m (0–25, 25–50, 50–75, 75–100, 100–150, 150–250, 250–500,
500–750, 750–1000, 1000–1500, 1500–2000, 2000–3000 m) at 5 sta­
tions
along
the
north–southtransect(29◦ 00′ N–42◦ 40′ N,◦′◦′
145 45 E− 149 40 E) from 19 October to 7 November 2017. Along thetransect, the NORPAC net equipped with a 100 μm mesh was also towed
vertically from 0 to 200 m at eight stations. VMPS samples were gently
filtered through a 50-μm mesh under a weak vacuum and then frozen
and stored at − 20 ◦ C. NORPAC net samples were gently sieved throughthree meshes (925 μm, 407 μm, and 112 μm) within a large bucket filledwith seawater. The zooplankton samples at each fraction were filteredonto a 50 μm mesh under a weak vacuum, then frozen and stored at− 20 ◦ C. For both cruises of Oshoro-Maru and Hakuho-Maru, watertemperature and salinity were measured by CTD (Sea-Bird Electronics,
Bellevue, USA) at each station, T-S diagrams were drawn, then the watermass classification was performed using the criteria of Anma et al.
(1990). For references on inorganic nitrogen, we provided the data as anelectronic supplement (Electronic Supplement 1).

the following equation,(1)

δ13C or δ15N = (Rsample/Rstandard − 1)
13

12

15

14

where, Rsample and Rstandard are the isotopic ratios ( C/ C or N/ N)of the sample and standard, respectively. The standards used were
Vienna Pee Dee belemnite and atmospheric nitrogen for carbon andnitrogen, respectively. The standard deviation of the repeated mea­
surements of multiple working standards was less than 0.2‰ for bothisotope ratios.
2.3. Statistical analysis
To clarify the effects of various factors (δ13C, sea area, depth, andsize) on the nitrogen stable isotopic ratio (δ15N), we created scatter plots
with δ15N as the dependent variable (Y) based on the data from eachcruise. For the data of the Oshoro-Maru cruises, the regression equation(2) was used for the two regions (subarctic and transitional domain),(2)

Y=aX+b15

13

where, Y is δ N (‰), X is δ C, and a and b are constants. To evaluate
the differences in the regressions between regions, an analysis ofcovariance (ANCOVA) was performed.
For the data based on VMPS samples from the Hakuho-Maru cruise,the vertical changes in δ15N (Y) were determined using the linear
regression equation,
Y = a X + b,(3)

where, X is the mid-point depth (m) of the sampling layer, and a and b
are fitted constants. Differences in the regressions between stations,were tested using ANCOVA. For the NORPAC net samples of the HakuhoMaru cruise, the linear regression equation (4) was used to analyse eachsize fraction (925 μm, 407 μm, 112 μm),2.2. Stable isotope analyses
In the land laboratory, samples were first freeze-dried, and thenoven-dried at 60 ◦ C for more than 24 h; subsequently, they were
powdered using a mortar and pestle and the, lipids were then extractedusing a 2:1 chloroform: methanol solution. After lipid extraction, thesamples were dried at 60 ◦ C for 24 h. The powdered samples were placedin a tin cup, and the stable isotope ratios of carbon and nitrogen were
measured using a continuous flow–isotope ratio mass spectrometer(Delta V Advantage, Thermo Fisher Scientific Inc., Waltham, MA, USA)
connected to an elemental analyser. The natural abundance of 13C or 15N
is expressed in per mil (‰) deviation from international standards using

Y=aX+b(4)

where, Y is δ15N, X is latitude (◦ N), and a and b are fitted constants.
Differences in the regressions were tested using by ANCOVA, with sizeand latitude as independent variables. Statistical analyses were per­formed using MS Excel or StatView (SAS Institute).

3

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Deep-Sea Research Part I 197 (2023) 104045

Fig. 2. Vertical and latitudinal changes in temperature (a), salinity (b), and T-S diagrams (c) based on the Oshoro-Maru cruise (left) and Hakuho-Maru cruise (right).
Differences in symbols denote stations in each cruise. Dashed lines represent the separation of the water masses (subarctic, transition domain, and subtropical region)(cf. Anma et al., 1990).

3. Results

and two geographically intermediate stations (37–39◦ N) forming thetransitional domain.

3.1. Hydrography

3.2. Regional differences in carbon and nitrogen stable isotope ratios

The vertical section contour plot and T-S diagrams of the watertemperature and salinity at each station for each cruise are shown in
Fig. 2. For the 155◦ E line observations by the Oshoro-Maru cruises in
May 2016, the sea surface temperature and salinity ranged from 3.0 ◦ C
to 18.1 ◦ C and from 33.0 to 34.7, respectively. The plot of each station in
the T-S diagram showed discontinuity around a salinity of 34.0 and was
divided into three stations with lower salinity at higher latitudes(42–44◦ N: subarctic region), and three stations with higher salinity atlower latitudes (37◦ 15′ N–40◦ 15′ N: transitional domain). For thenorth–south observation line by the Hakuho-Maru in October 2017, theplotted areas of the deep layer in the T-S diagram showed similar tem­
perature and salinity values for all stations. On the contrary, the plotted
areas of sea surface waters in the T-S diagram were largely divided into
three groups: two stations with cold and fresh waters (42–44◦ N, sub­arctic), four stations with hot and saline waters (29–35◦ N, subtropical),A scatter plot of the stable isotope ratio between δ15N and δ13C of thesamples collected in the subarctic region of the Oshoro-Maru cruise isshown in Fig. 3. We found that δ13C ranged from − 25.5‰ to − 19.0‰
and δ15N ranged from 3.2‰ to 12.0‰, and a significant linear regression
was obtained between them; Δδ15N/Δδ13C was 1.210 (r2 = 0.76, P <0.0001).
A scatter plot of δ15N and δ13C in the transitional domain is shown in
Fig. 4. The plotted areas of δ13C and δ15N varied greatly from those of
the subarctic region described above, with the plotted areas of δ13C on
the right (high δ13C values) and those of δ15N starting at a lower δ15N
value. A significant linear regression between δ13C and δ15N was also
obtained for the transitional domain, with Δδ15N/Δδ13C being 2.112 (r2= 0.650, P < 0.0001).
The seven species that commonly occurred in both areas were the4

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Deep-Sea Research Part I 197 (2023) 104045

Fig. 3. Scatter plots between δ15N (Y) and δ13C (X) for various pelagic organisms collected at the epipelagic level (0–150 m) of the western subarctic Pacific(42–44◦ N, 155◦ E) during 14–17 May 2016. Symbols and bars denote means and standard deviations, respectively. The regression line was analysed based on thewhole dataset plotted in the panel.

Fig. 4. Scatter plots between δ15N (Y) and δ13C (X) for various pelagic organisms collected at the epipelagic level (0–150 m) of the transitional domain of the western
North Pacific (37◦ 15′ –40◦ 15′ N, 155◦ E) from 14 to 17 May 2016. Symbols and bars denote means and standard deviations, respectively. The regression line wasanalysed based on the whole dataset plotted in the panel.

copepods: Euchirella rostrata (Claus 1866), Metridia pacifica Brodsky
1950, Neocalanus cristatus (Krøyer 1848), and N. plumchrus (Marukawa
1921); the euphausiid: Euphausia pacifica Hansen 1911; the squid:
Gonatopsis borealis Sasaki 1923, and the fish: Oncorhynchus gorbuscha(Walbaum 1792) (Figs. 3 and 4). A common pattern between regions
was observed in the stable isotope ratios of these species. Thus, high
δ15N and low δ13C in the subarctic region and low δ15N and high δ13C inthe transitional domain were common patterns observed across the taxa
(Table 1).
ANCOVA for the linear regressions between δ13C and δ15N in thesubarctic and transitional domains showed a significant interaction be­
tween area and δ13C (P < 0.01, Table 2), indicating that the regression

equation in both areas varied. Comparing the linear regressions for bothregions, the zooplankton value at the lower left end of the regression
varied substantially, whereas the values of the fish and squid at theupper right end of the regression were similar (Figs. 3 and 4). Thus, thedifferences in the regressions could be derived from the differences inzooplankton values.
3.3. Vertical changes in δ13C and δ15N of zooplankton
Vertical changes in δ13C and δ15N of zooplankton down to a depth of3000 m at five stations obtained during the cruise of Hakuho-Maru areshown in Fig. 5. The δ13C values showed large variations in the5

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Deep-Sea Research Part I 197 (2023) 104045

Table 1
Regional comparison of δ15N and δ13C of the commonly occurring species in the subarctic (SA: 42–44◦ N) and transitional domains (TR: 37◦ 15′ –40◦ 15′ N) along 155◦ Eof the western North Pacific from 14 to 20 May 2016. Values are means ± standard error. The numbers of measurements are shown in parentheses. Regional differenceswere tested by Mann-Whitney U test. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ns: not significant, -: not applicable. TL: total length, ML: mantle length, FL: fork length.
TaxaSpecies/stage or length
Copepoda
Euchirella rostrata
C6F
Metridia pacifica
C6F
Neocalanus cristatus
C5
Neocalanus plumchrus
C5
Euphausiacea
Euphausia pacifica
TL: 4–5 mm
Cephalopoda
Gonatopsis borealis
ML: 152–200 mm
Salmonidae
Oncorhynchus gorbuscha
FL: 340–435 mm

δ15N

δ13C

SA

TR

U testSATR

U test3.90(1)4.76 ± 0.53
(4)
5.21 ± 0.39
(19)
3.63 ± 0.24
(6)3.00 ± 0.96
(6)
4.10 ± 1.32
(3)
3.09 ± 0.64
(8)1.70 ± 0.32
(14)–

− 24.89(1)− 24.75 ± 0.28
(4)
− 24.26 ± 0.49
(19)
− 25.62 ± 0.84
(6)

− 21.10 ± 0.77
(6)
− 21.27 ± 0.78
(3)
− 22.43 ± 0.31
(8)
− 22.36 ± 0.74
(14)–

4.37 ± 0.66
(3)

3.52 ± 0.52
(7)ns

− 23.44 ± 0.76
(3)

− 20.99 ± 0.96
(7)

*

11.98
(1)

11.52 ± 0.37
(5)–

− 19.07(1)− 19.06 ± 0.11
(5)–

10.46 ± 0.68
(15)

9.49 ± 0.64
(3)*

− 21.33 ± 0.64
(15)

− 21.27 ± 0.64
(3)

ns

ns
**
**

df

SS

F-value

P

1
1
1
192

71
1586
126
987

13.9
308.7
24.4

***
****
****

*
***

of δ15N were observed for regressions of the largest (925 μm), inter­mediate (407 μm), and smallest size classes (112 μm), respectively. The
differences in δ15N between the size classes were at a factor of
1.06–1.49. ANCOVA showed no significant differences in size or size ×
latitude interactions (Table 4), indicating that the linear regressions forthe three size classes could be treated as the same line.

Table 2
Results of ANCOVA for δ15N of the various pelagic organisms collected byplankton net tows and gillnet surveys in the subarctic and transitional domain of
the western North Pacific from 14 to 20 May 2016. For this analysis, area andδ13C were applied as independent variables. df: degrees of freedom, SS: sum ofsquares, ***: P < 0.001, ****: P < 0.0001.
Area
δ13C
Areaｘδ13C
Error

**

4. Discussion
4.1. Regional differences in Δδ15N/Δδ13C
In this study, significant linear regressions were observed between
δ15N and δ13C in both the subarctic and transitional domains, whichreflect the high correlations of the stable isotope ratios in each region(Figs. 3 and 4). This indicates the trophic accumulation effects of 15N(Kline, 1999; Hobson et al., 2002), and the single carbon source of the
primary products supporting marine ecosystems (Fanelli et al., 2011;
Williams et al., 2014). In general, low δ15N–δ13C correlations have beenreported for marine ecosystems where the original primary production
sources have various origins, such as marine snow, phytodetritus, re­generated POM, and the effects of river plumes (Fanelli et al., 2011).
Seasonal differences also affect the correlations between δ15N and13
δ C (Aita et al., 2011). High δ15N–δ13C correlations were found forpulses from fresh organic matter based on primary production byphytoplankton blooms, and high δ13C of zooplankton was observed
when the surface chlorophyll-α concentration was high (Fanelli et al.,2011). In the eastern subarctic Pacific, there are seasonal changes in thecorrelation coefficients of the δ15N–δ13C regressions of the dominantlarge-sized copepod N. cristatus, and their isotope ratios are reported tobe divergent after the end of the spring phytoplankton bloom (Kline,1999). As the present study was conducted in May with high phyto­plankton productivity in the western North Pacific (Longhurst, 2007),
high correlation coefficients between the stable isotope ratios were ob­tained for both the subarctic and transitional region. While the re­gressions between δ15N and δ13C of various taxonomic groups(copepods, euphausiids, amphipods, and chaetognaths) were significant
from March–October, substantial changes in the slopes of the regressions
were reported within the 2-month intervals (Aita et al., 2011).
As notable characteristics observed in this study, greater differencesin the linear regressions for δ15N and δ13C were present between thesubarctic and transitional domains even within the same season (Fig. 3

epipelagic zone (0–200 m), but settled around − 21 to − 19‰ at depths
below 200 m, showing little difference with depth and between stations.
In contrast, the δ15N showed lower values near the sea surface andincreased with increasing depth, especially for the three stations in thesubtropical region (29◦ , 31◦ and 33◦ N), where significant linear re­
gressions were observed. The significance of vertical changes in δ15N
with increasing depth was more pronounced at lower latitudes (29◦ N: r2= 0.698, P < 0.001), moderate at higher latitudes (33◦ N: r2 = 0.495, P <0.05) and intermediate between the two stations (31◦ N: r2 = 0.668, P <0.01). There were no significant vertical changes in δ15N with depth in
the transitional (37◦ N) and subarctic (41◦ N) domains. Especially at
41◦ N, there were few vertical changes in δ15N from the surface to adepth of 3000 m.
ANCOVA was performed for the linear regressions obtained for thethree stations in the subtropical region. Although a difference by lati­
tude (P < 0.05) was observed, no interactions were detected for latitude
× depth (Table 3). These results indicate that the intercepts of the re­gressions for δ15N, at the surface layer varied between the stations, while
their slopes remained the same.
3.4. Latitudinal and size changes in stable isotopes of zooplankton
Latitudinal changes in the stable isotopes of the size-fractionatedzooplankton samples at 0–200 m depth during the cruise of the
Hakuho-Maru are shown in Fig. 6. For δ13C, no variations with latitude
or size were observed. In contrast, for δ15N, increases with increasing
latitude were observed for all size classes (r2 = 0.787–0.826, P < 0.001).
Among the size classes, the largest, intermediate, and smallest values
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Deep-Sea Research Part I 197 (2023) 104045

Fig. 5. Vertical changes in δ13C (left) and δ15N (right) of mesozooplankton down to a depth of 3000 m collected by 63-μm mesh VMPS at five latitudinal stations in
the western North Pacific from 25 October to November 7, 2017. Linear regressions were analysed for each station and dashed lines showed those showing asignificant relationship.

warming and start of the phytoplankton bloom in the transitional
domain. These hydrographic characteristics may correspond to thecondition in June reported by Aita et al. (2011). Such latitudinal andseasonal variations in stable isotope ratios were observed for species that
occurred in both regions, especially for zooplankton species (Table 1).
Our study’s treatment of Δδ15N/Δδ13C values adds to the data of Aita
et al. (2011) and, Kobari et al. (2022) from the same region. For com­
parison, the regressions between δ15N and δ13C from these two studies
and those from our study are shown in Fig. 7. From this figure, it isrecognised that the plotted areas of this study were broader than those of
the other two. This is partly because the previous studies included only
the zooplankton, while our study included organisms ranging fromzooplankton to the higher trophic levels, such as fishes and squids
collected by gillnetting. Compared to those of previous studies, the re­gressions of the subarctic and transitional domains in this study are close
to those of St. K2 in the subarctic and St. S1 in the subtropical regions by
Kobari et al. (2022), respectively. Common patterns observed in bothstudies are that δ13C has high values at low latitudes, whereas δ15N has
high values at high latitudes (Fig. 7). It is also notable that such regional
differences in the stable isotope ratios were mostly caused byzooplankton, and few regional differences were observed in the higher
trophic level organisms mentioned. These facts (small regional differ­ences in higher trophic levels) are considered to be a reflection of thegeneration length of each taxon. For shorter-lived zooplankton, theirisotope ratios would have large fluctuations that vary with region andseason, whereas those of fishes and squids would be more stable aroundthe values mentioned before (δ15N: δ13C = 1.6) in both regions, because
they have comparatively long generation lengths.
Higher δ13C values of zooplankton have been attributed to the faster
growth rate of the phytoplankton community (Pomerleau et al., 2014;
Henschke et al., 2015). In the northern Gulf of Alaska, the δ13C of
N. cristatus is reported to be lower in different seasons and regions,characterised by smaller phytoplankton cell sizes (Kline, 2009). Phyto­plankton growth rates are positively affected by, higher water temper­
atures (Sherman et al., 2016). As the water temperature was high in thetransitional domain (Fig. 2), the growth rate of the phytoplankton
community is expected to be faster than that of the subarctic region,
which would be expected to produce high δ13C values for

Table 3Results of ANCOVA for δ15N of zooplankton samples collected by VMPS to adepth of 3000 m at five stations along a north–south transect (29–41◦ N) in thewestern North Pacific. For this analysis, latitude and depth were applied as in­dependent variables. df: degrees of freedom, SS: sum of squares, *: P < 0.05,****: P < 0.0001, ns: not significant.
Latitude
Depth
LatitudeｘDepth
Error

df

SS

F-value

P

2
1
2
27

12.4
61.0
6.4
37.1

4.54
44.34
2.32

*
****
ns

and, 4). The presence of common regressions for δ15N and δ13C ofzooplankton has been reported in four different oceanic regions(Southern Ocean, Oyashio, Gulf of Alaska, and the warm-core ring off
Sanriku) (Aita et al., 2011). For this reason, as the amino acid metabolic
systems are identical throughout the species and regions, shared
Δδ15N/Δδ13C values of approximately 1.6 are present across the regions(Wada and Noguchi, 2017). In this study, the Δδ15N/Δδ13C values in thesubarctic and transitional domains were 1.210 and 2.112, respectively,
which varied substantially from the reported values (1.6). According to
Aita et al. (2011), Δδ15N/Δδ13C values fluctuate seasonally, even within
the same region. For example, they reported the same Δδ15N/Δδ13C
values (1.23) in March and October, 0.61 in May and 1.39 in June forzooplankton in the Oyashio region. These results suggest thatΔδ15N/Δδ13C values vary greatly from May to June, during this region’s
productive season. As the present study was also conducted in May, thepresence of large spatio-temporal variations in Δδ15N/Δδ13C corre­
sponded well with the results of Aita et al. (2011). According to Aita
et al. (2011), water temperature increased and nitrate concentrations
decreased from May–June, and subsequently, zooplankton δ15N
decreased and δ13C increased simultaneously. While the actual values
were varied, the results of Aita et al. (2011) in May (low Δδ15N/Δδ13C)
matched the results of the cold subarctic region of this study closely. In
contrast, the results of June (high Δδ15N/Δδ13C) in Aita et al. (2011)were consistent with the results of the warm transitional domain in thisstudy (Figs. 3 and 4). These similarities may be due to the earlier onset of7

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Deep-Sea Research Part I 197 (2023) 104045

phytoplankton, consequently inducing high δ13C values in zooplankton.
For the δ15N of the large copepods Neocalanus spp., predation pres­sure is high under cold conditions because of the weak development of
the thermocline, which may present phytoplankton blooms, and induce
the dominance of microbial organisms. This has been proposed as an
explanation for high δ15N values in Neocalanus spp. under cold condi­
tions before (Chiba et al., 2012). The nitrate concentration also affects
δ15N (Chiba et al., 2012; Pomerleau et al., 2014). Thus, low nitrateconcentrations and high δ15N values may enhance the uptake of heavier
δ15N by the phytoplankton, increasing δ15N of food phytoplankton andalso for Neocalanus (Chiba et al., 2012; Pomerleau et al., 2014). Thus,
the high δ15N and low δ13C values observed in the subarctic region inthis study may be due to differences in phytoplankton growth rates
caused by cold water temperatures. Temperature conditions strongly
affected both δ13C and δ15N values. In this study, while significant re­
gressions were observed for both the subarctic and transitional domain,the Δδ15N/Δδ13C values varied substantially (1.210 and 2.112, respec­tively, Figs. 3 and 4) from the theoretical value (about 1.6, Wada andNoguchi, 2017). This is attributed to changes in the stable isotope ratiosof the organisms in response to environmental changes mainly due to thewater temperature (Fig. 2) and nitrate concentration in each region(Electronic Supplement 1).
4.2. Vertical changes in stable isotope ratios
In the present study, there was no significant relationship between
δ13C and depth at all stations (Fig. 5). Such phenomena have been re­ported in studies of various zooplankton taxa from depths of 0–2500 m
in the north–eastern Pacific off the coast of Vancouver (Burd et al.,Table 4
Results of ANCOVA for δ15N of zooplankton samples collected by NORPAC net
from depths of 0–200 m at eight stations along the latitudinal transect(29◦ 00′ N–42◦ 40′ N) in the western North Pacific. For this analysis, size andlatitude were applied as independent variables. df: degrees of freedom, SS: sumof squares, ****: P < 0.0001, ns: not significant.

Fig. 6. Latitudinal changes in δ13C (upper) and δ15N (lower) of sizefractionated (925, 407, and 112 μm) mesozooplankton samples collected by
150-μm mesh NORPAC net at depths of 0–200 m between 29◦ 00′ –42◦ 40′ N,
147◦ 12′ –145◦ 44′ E in the western North Pacific Ocean from 25 October toNovember 7, 2017. Linear regressions were analysed for each size fraction anddashed lines indicate significant sizes (P < 0.05).

Size
Latitude
SizeｘLatitude
Error

df

SS

F-value

P

2
1
2
21

0.53
64.36
0.24
15.45

0.36
87.52
0.16

ns
****
ns

Fig. 7. Inter-study comparison of the regressions between δ15N and δ13C evaluated during various studies in the western North Pacific Ocean. Comparable data forthe present study were sourced from Aita et al. (2011) and Kobari et al. (2022), which were also conducted in this region. For sampling locations of each study,
see Fig. 1.
8

D. Kim et al.

Deep-Sea Research Part I 197 (2023) 104045

2002), zooplankton between 0 and 2000 m in the eastern Atlantic Ocean
at 37◦ N to 21◦ S (Bode et al., 2015), and zooplankton at 0–1200 m in theeastern tropical North Pacific (Williams et al., 2014). In this study, large
fluctuations were observed in δ13C at shallower depths of 0–200 m(Fig. 5). A similar situation (large fluctuations in δ13C values at shal­
lower depths) was reported off the coast of Vancouver (Burd et al.,2002). The high δ13C value, the high growth rate of phytoplankton has
been reported as the cause of high δ13C values (Pomerleau et al., 2014;
Henschke et al., 2015). Thus, the high fluctuations in δ13C values in thesurface layer are thought to reflect spatiotemporal changes in the growthrate of phytoplankton. In contrast, in the deep layer (>200 m), the δ13C
value was in a narrow range (− 21 to − 19) and did not change vertically(Fig. 5). This is thought to indicate that the labile component of the POM
decomposes first, then the persistent components remain in the deep
layers and settle, resulting in the constant stable isotope values of δ13C inthe deeper layer (Burd et al., 2002). Thus, the δ13C value can fluctuate at
the sea surface layer where phytoplankton actively reproduce, while inthe deep layer, δ13C likely remains at constant values throughout thelayer. This is because of the distance separating it from the sea surface,where primary production occurs, and the aging of the utilisation of thematerial fluxes may occur, causing stabilization of the δ13C values.
A trend of increasing zooplankton δ15N values with increasing depth
was observed in the subtropical region of this study (Fig. 5). The same
trend is well known to occur at the lower latitudes in the temperate totropical regions, including depths of 0–4200 m in the Mediterranean Sea(Koppelmann et al., 2003, 2009), 0–2000 m depths at 37◦ N–21◦ S in theeastern Atlantic Ocean (Bode et al., 2015), and 0–1200 m in the tropical
Eastern North Pacific (Williams et al., 2014). For the western North
Pacific, the depth-increasing trend of δ15N has also been reported in thesubarctic and subtropical regions (Kobari et al., 2022). The verticalchange in POM is considered to be a factor causing the increase in δ15N
with increasing depth. Thus, during the biodegradation of POM by mi­
crobial consumption, bonds containing 14N are preferentially broken,
and the remaining isotopically heavier δ15N connection results in higher
δ15N in POM in the deep layer (Koppelmann et al., 2009; Laakmann and
Auel, 2010; Richards et al., 2020). Consumption of POM by zooplankton
in the mesopelagic zone, repacking of POM by fecal pellet egestion, andits coprophagy also contribute to the higher δ15N in the deeper layer(Williams et al., 2014; Richards et al., 2020). As depth increases may
induce additional feeding and predation by various zooplankton, theδ15N values of zooplankton in the deep sea are thought to be higher thanthose near the sea surface (Bode et al., 2015). Thus, the increasing δ15N
values with increasing depth observed in the subtropical region arethought to be caused by the repacking of POM that, originated from thesea surface layer, during sinking to the deep layer.
In contrast, no vertical changes in δ15N values were observed in thetransitional domain and subarctic regions (Fig. 5). It should also be
noted that the significance of the regressions between δ15N and depth at
the three stations in subtropical regions was low at higher latitudes. In
the western subarctic Pacific, large-sized copepods (Neocalanus spp. andEucalanus bungii Giesbrecht 1893) that perform seasonal ontogenetic
vertical migration to the deep, considered a diapause phase in their life
cycles, dominate the zooplankton biomass (Ikeda et al., 2008). As the
sampling in this study was conducted from October–November, when
the copepods migrate down to the deep sea to start diapause, they are
estimated to contribute 8–67% (mean ± SD: 31 ± 19%) of the total
zooplankton biomass between depths of 200 and 3000 m in the western
subarctic Pacific during summer (Yamaguchi et al., 2004). The domi­nance of the diapausing copepods was also the case for the deep layer
samples in the subarctic and transitional regions (Kim, unpublisheddata). As feeding of these copepods occurs at the sea surface layer anddoes not occur in the deep layer (Tsuda et al., 2004), their δ15N values
are expected to be similar throughout the layers. It should also be notedthat the diapausing copepods are transported southwards by submerged
Oyashio water, and their occurrence in the deep layer extends to
1000–2000 m at 30◦ N (Yamaguchi et al., 2004; Kobari et al., 2008). Dueto the large-body sizes of subarctic copepods, their occurrence in the
deep layer only may affect the δ15N value of the transitional domain andnorthern subtropical regions, which may reduce the significance of theδ15N regressions with increasing depth. Thus, the decreasing signifi­
cance of the regressions between δ15N and depth at the higher latitudes
is thought to be due to the dominance of these large subarctic copepods
in the deep layer.
4.3. Latitudinal and size changes in stable isotopes
For all organism sizes, we found no significant latitudinal changes inδ13C values (Fig. 6). Such results have been reported for zooplankton
from various oceanic regions, including the Southern Ocean, Oyashio,
Gulf of Alaska, and warm-core rings off Sanriku (Aita et al., 2011), andin the southern Indian Ocean (Richoux and Froneman, 2009). For co­
pepods, no latitudinal changes in δ13C have been reported in the eastern
Atlantic Ocean (Bode et al., 2015), and the Greenland and Barents Seas
(Sato et al., 2002). No differences in δ13C with size were reported in astudy on capelin in the Arctic Ocean (Ogloff et al., 2020). A reason forthe lack of latitudinal and size changes in δ13C may lie in the almost
identical major metabolic pathways such as amino acid synthesis in
animals resulting in the absence of differences in stable isotope ratioseven in different regions (Aita et al., 2011), in addition to the effects ofcarbon sources. For example, there are no changes in the δ13C value of
copepods at high latitudes during the winter season because they feed
primarily during a phytoplankton bloom from spring to summer and do
not feed during the rest of the year. Consequently, their δ13C values arestable, because they derived from the same phytoplankton carbon
sources (Sato et al., 2002). In this study, sampling was not conductedduring phytoplankton bloom season, as the samples were collected at
depths of 0–200 m, thus, they may not have contained diapausing co­
pepods. According to Richoux and Froneman (2009), the δ13C values of
euphausiids, amphipods, and copepods were not related to externalvariables (salinity, temperature, chl. a, size, etc.). Thus, there are many
examples showing that δ13C values do not change with region, corre­
sponding with the pattern in this study.
In this study, across all size classes, the δ15N value was higher athigher latitudes (Fig. 6). This patterns in δ15N of various zooplankton
taxa have been reported in the eastern tropical North Pacific (Williams
et al., 2014) and eastern Mediterranean Sea (Koppelmann et al., 2003,
2009). As reason for this pattern of δ15N (low at low latitudes) is partly
due to the nitrogen fixation ability of phytoplankton. Thus, cyanobac­
teria, having a nitrogen fixation ability with a low δ15N value (1–4‰)
among the epipelagic plankton of the eastern Mediterranean, caused byatmospheric nitrogen, is the dominant primary producer and respon­sible for the extremely low δ15N baseline in the region (Koppelmann
et al., 2003, 2009). The abundance of cyanobacteria is high at lower
latitudes (Shiozaki et al., 2015; Wang et al., 2021). These factors induce
low δ15N values in zooplankton at lower latitudes. Furthermore, a low
nitrate concentration is available (Electronic Supplement 1). As 14NO−3 isabundant and is utilized preferentially by phytoplankton, incomplete
utilisation of nitrate creates POM with low a δ15N values (2–3‰) in thetropical regions (Williams et al., 2014). In the sea off Western Australia,
the cold upwelled water contains a high δ15N value because of thedominance of upwelled nitrate in the nutrients available for primary
production, while a low δ15N occurs in the normal warm region, which isdue to the dominance of regenerated nitrogen sources for primary pro­duction (Waite et al., 2007). Such latitudinal changes in nitrate con­centrations are also present in the western North Pacific (Shiozaki et al.,2015; Wang et al., 2021; Electronic Supplement 1), where the present
study was conducted.
The δ15N value is known to increase by approximately 3.4‰ byincreasing one trophic level, thus indicating their trophic level in eachecosystem (Minagawa and Wada, 1984). In the present study, the δ15N
value of zooplankton was higher for larger organism size classes.
However, the differences in δ15N values with size were less than 2‰
9Deep-Sea Research Part I 197 (2023) 104045

D. Kim et al.

(Fig. 6), indicating that the differences could not have been caused bytrophic level differences. Generally, it is well known that the small-sizedorganisms tend to be herbivores with low δ15N values, whereas
large-sized organisms tend to be carnivores with high δ15N values(Koppelmann et al., 2003; Richoux and Froneman, 2009; Hiwatari et al.,2011). Within the same species, it has also been reported that the dietshifts to carnivory as the organism grows, inducing higher δ15N values inolder specimens (Hop et al., 2006; Waite et al., 2007; Ogloff et al.,2020). Common to all size classes, a significant linear regression be­
tween the δ15N values and latitude was observed, with δ15N increasing
with increasing latitude (Fig. 6). The subsequent ANCOVA revealed nointeractions with size, or size × latitude (Table 3), indicating that the
regressions observed for the three size classes can be treated as the same.
Thus, within the taxonomic category of zooplankton, differences in δ15N
values are much greater for latitudinal changes than those caused by thedifferences in sizes.

improve the manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dsr.2023.104045.
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5. Conclusion
Here, we conducted three analyses on the stable isotope ratios (δ15Nand δ13C) of zooplankton in the western North Pacific. Different Δδ15N/
Δδ13C values were obtained for the subarctic and transitional domains,which could be attributed to changes in the zooplankton community
composition, to species with shorter generation lengths. For verticalchanges, the significance of regressions increased with increasing depth,
but only in the subtropical regions. The cause of the absence of verticalchanges in δ15N values in the subarctic region may be the dominance oflarge-sized interzonal copepods that perform an ontogenetic verticalmigration from the sea surface to the deep sea to undergo diapause in
their life cycle. Thus, the dominance of interzonal copepods in thezooplankton biomass may mask the vertical changes in their δ15N
values. On the contrary, the scarcity of such interzonal copepods in thesubtropical region caused an accumulation of the effect of repacking byzooplankton on the sinking particles, especially for the deeper layer,
which may have induced higher δ15N values here. The δ15N ofzooplankton in the size fraction was higher for the larger size classes, but
the differences in δ15N caused by size were much smaller than thosecaused by latitudinal changes. A high δ15N value of zooplankton at
higher latitudes was observed for all size classes. As a limitation of thisstudy, since this study conducted only one each occasion or year, yearlychanges in the stable isotope ratios could not be evaluated. For future
study, evaluation of such issues (yearly changes) would be needed.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
We would like to thank the crew members of T/S Oshoro-Maru and R.
V. Hakuho-Maru for their assistance in collecting the samples and CTD
data. This work was supported by the Arctic Challenge for Sustainability
II (ArCS II) Program (Grant Number JPMXD1420318865), Environment
Research and Technology Development Fund (JPMEERF20214002) ofthe Environmental Restoration and Conservation Agency of Japan; andGrants-in-Aid for Challenging Research (JP20K20573 [Pioneering]) and
Scientific Research (JP22H00374 [A], JP20H03054 [B], JP19H03037[B], JP17H01483 [A], and JP16H02947 [B]) from the Japanese Society
for the Promotion of Science (JSPS). We would also like to thank thethree anonymous reviewers for their constructive inputs that helped
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