TYPE Original Research
PUBLISHED 20 September 2023
DOI 10.3389/fmars.2023.1168015

OPEN ACCESSEDITED BY

Guang Yang,
Chinese Academy of Sciences (CAS), China
REVIEWED BY

Daria Martynova,
Zoological Institute (RAS), Russia
Elizaveta Ershova,
UiT The Arctic University of Norway,
NorwayVladimir G. Dvoretsky,
Murmansk Marine Biological Institute,
RussiaEmilia Trudnowska,
Polish Academy of Sciences, Poland

Geographic variation in
population structure and grazing
features of Calanus glacialis/
marshallae in the Paciﬁc
Arctic Ocean
Minami Ishihara 1*, Kohei Matsuno 1,2, Koki Tokuhiro 3,
Yasuhiro Ando 1, Kazutoshi Sato 4 and Atsushi Yamaguchi 1,2
Faculty/Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan,
Arctic Research Center, Hokkaido University, Sapporo, Hokkaido, Japan, 3 Demonstration Laboratory,
Marine Ecology Research Institute, Kashiwazaki, Niigata, Japan, 4 Arctic Environment Research Center,
National Institute of Polar Research, Tachikawa, Tokyo, Japan
1

2*CORRESPONDENCE

Minami Ishihara
minami0525konakichi@eis.hokudai.ac.jp
RECEIVED 17 February 2023
ACCEPTED 23 August 2023
PUBLISHED 20 September 2023
CITATION

Ishihara M, Matsuno K, Tokuhiro K, Ando Y,
Sato K and Yamaguchi A (2023)
Geographic variation in population
structure and grazing features of Calanus
glacialis/marshallae in the Paciﬁc
Arctic Ocean.
Front. Mar. Sci. 10:1168015.
doi: 10.3389/fmars.2023.1168015
COPYRIGHT© 2023 Ishihara, Matsuno, Tokuhiro, Ando,
Sato and Yamaguchi. This is an open-accessarticle distributed under the terms of theCreative Commons Attribution License(CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and thatthe original publication in this journal iscited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does notcomply with these terms.

Calanus glacialis/marshallae is a dominant zooplankton species in the Paciﬁc Arctic
Ocean that is widely distributed in shelf areas, and it plays a vital role in connecting
primary production to higher trophic levels. Its phenology is well adapted tohydrography, but there is little available information about regional and dielchanges in population structure and grazing features. In this study, weinvestigated C. glacialis/marshallae during autumn 2019 in the Eastern andNortheastern Chukchi and Canadian basins to reveal geographic and dielvariations in population structure, body size, grazing activity, and fatty acidcomposition. The abundance of C. glacialis/marshallae was found to be high on
the slopes and low on the shelves. Body size (prosome length) was well described by
the Bě lehrá dek equation combined with in-situ temperature throughout the
sampling region. Cluster analyses based on hydrographic parameters were divided
into four regions: southern shelf, northern shelf, slope, and basin. The southern shelf
was dominated by copepodite stage ﬁve (C5) transported from the Bering Sea by
Paciﬁc waters. C4 and C5 were dominant on the northern shelf, suggesting that they
grew slower than those on the southern shelf, and the populations also exhibited
higher concentrations of fatty acids originating from dinoﬂagellates than thoseoriginating from the pan-Arctic Ocean, indicating low productivity in the region.
The population on the slope had the highest abundance, C4 was dominant, andlarge amounts of diatom-derived eicosapentaenoic acid (EPA). These features are
attributed to the upwelling of populations and nutrients that support diatom growth.
In the basin, the early copepodite stages of composition were distinctly higher thanthose recorded in previous studies, because larger amounts of organisms ﬂow into
the region, resulting in more extended reproduction periods. In the basin, small andlarge forms of C5 were simultaneously found, and the small form exhibited a dielgrazing activity pattern, but the large forms did not. These ﬁndings suggest their well
adaptation in changing of the Paciﬁc Arctic Ocean.
KEYWORDS

Calanus glacialis/marshallae, Paciﬁc Arctic Ocean, population structure, body size, fatty
acid, gut pigment

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species exhibits plasticity in response to environmental conditions
that are reﬂected in the biological features of the species, such as
body size, grazing activity, and the accumulation of fatty acids.
The body size of copepods varies depending on the temperatureand prey conditions (Digby, 1954; Viitasalo et al., 1995; Kobari
et al., 2003). Low temperatures and high food availability stabilize
the growth of copepods (Vidal, 1980), which increases the body size
(Viitasalo et al., 1995). Geographic variations in the body size of thisspecies have been reported in the Atlantic sector of the Arctic Ocean
when conducting interspeciﬁc comparisons with Calanus
ﬁnmarchicus (Kosobokova, 1999; Choquet et al., 2018), but little
information is available for the Paciﬁc Arctic Ocean (Ashjian
et al., 2003).
Calanus glacialis is omnivorous, feeds mainly on diatoms anddinoﬂagellates (Søreide et al., 2008; Yeh et al., 2020), and adapts
ﬂexibly to changes in its prey composition (Stevens et al., 2004a;
Banas et al., 2016; Freese et al., 2016). With respect to feeding, thefatty acid composition has been well analyzed, mainly in the
Atlantic sector of the Arctic Ocean (Stevens et al., 2004a; ElSabaawi et al., 2009; Falk-Petersen et al., 2009; Trudnowska et al.,2020). The fatty acid composition of zooplankton varies with diet
(Stevens et al., 2004b). Zooplankton contains unique fatty acids thatare taken up and preserved without chemical changes in the body;fatty acids can be analyzed to obtain an index of their feeding
records (El-Sabaawi et al., 2009). For example, diatoms are rich ineicosapentaenoic acid (EPA), and dinoﬂagellates are rich indocosahexaenoic acid (DHA; Budge and Parrish, 1998). The gut
pigment is another index of the feeding activity of copepods, and it
is useful for evaluating diel changes in grazing activity (Conover andHuntley, 1991); however, it has also not been well studied in the
Paciﬁc Arctic Ocean (Matsuno et al., 2015). Therefore, the regional
differences in body size, feeding activity (fatty acid composition),and diel grazing patterns during autumn remain unknown. In thisstudy, therefore, we investigated the population structure andecological features (body size, feeding activity, and fatty acidcomposition) of C. glacialis to assess its current state in thechanging Arctic Ocean.

1 Introduction
Calanus Leach, 1816 is the dominant copepod genus in thezooplankton biomass within the Paciﬁc Arctic Ocean (Kosobokova
and Hirche, 2009). Three Calanus species have been identiﬁed indifferent areas of the Paciﬁc Arctic Ocean: Calanus hyperboreus in
the basin (Conover, 1988), Calanus glacialis on the shelf(Kosobokova and Hirche, 2009; Kosobokova and Hopcroft, 2010),
and Calanus marshallae in the southeastern Bering Sea (Frost, 1974;
Vidal and Smith, 1986). Of these, C. hyperboreus has the largest
body size and can be easily distinguished from the other two species.
Calanus glacialis and C. marshallae are morphologically similar anddifﬁcult to distinguish (Frost, 1974), although they have been
distinguished by genetic analysis (haplotype-based on 18S rRNA),and results have indicated that C. marshallae is transported to theshelf by the Paciﬁc waters (Nelson et al., 2009; Ashjian et al., 2021).
However, a genetic analysis was not conducted in the current study,
and the C. glacialis/marshallae species complex is therefore referred
to as C. glacialis in this work. Calanus glacialis has two populations(the Bering Sea and Arctic Basin populations that can also be
distinguished using haplotype analyses (Nelson et al., 2009; Ashjian
et al., 2017; Pinchuk and Eisner, 2017; Ashjian et al., 2021).
Although these populations are believed to be morphologicallyindistinguishable, haplotype analysis indicates that the Bering Sea
population is distributed on the Chukchi Sea shelf, and the Arctic
Basin population is distributed north of the Beaufort Sea slope/
Basin (Nelson et al., 2009). In addition, these populations appear todiffer in their copepodid stage composition during the same season(Ershova et al., 2015; Pinchuk and Eisner, 2017), suggesting that thelife cycles differ between populations.
Calanus glacialis is the dominant biomass species on the shelf of
the Paciﬁc Arctic Ocean (Kosobokova and Hirche, 2009;
Kosobokova and Hopcroft, 2010), and it plays an essential role inconnecting primary production to higher trophic levels in ﬁsh,birds, and mammals (Dickson and Gilchrist, 2002; Bengtson et al.,2005; Falk-Petersen et al., 2009; Crawford et al., 2012; Choquet
et al., 2018). The population peaks near the shelf border (the shelf
break) and declines sharply in the basin (Kosobokova and Hirche,
2009; Wassmann et al., 2015; Ershova et al., 2021). Calanus glacialis
has a generation length of 1–3 years (Kosobokova, 1999; Ashjian
et al., 2003; Falk-Petersen et al., 2009), and it grows from an egg to
C3 and C4 within one year (Scott et al., 2000). The species has
mixed reproductive strategies depending on food conditions(Plourde et al., 2005; Daase et al., 2013): capital breeding (basedon internal energy storage before the onset of the spring bloom) andincome breeding (utilizing ice algal or pelagic blooms) (Varpe et al.,2009). Due to this ﬂexibility, their reproduction is initiated under
sea ice and continues for one to two months using ice algae andpelagic phytoplankton blooms (Tourangeau and Runge, 1991;
Campbell et al., 2009; Daase et al., 2013). However, if individuals
reproduce in the late season, they cannot grow to the diapause stage
in a short period (Falk-Petersen et al., 2009; Daase et al., 2013). As
the developmental time between the stages is negatively related totemperature under sufﬁcient food conditions (Corkett et al., 1986;
McLaren et al., 1988), it is acknowledged that the life cycle of this

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2 Materials and methods
2.1 Field survey and onboard analysis
Zooplankton sampling was conducted via the R/V Mirai in the
Paciﬁc Arctic Ocean (66.50–78.04˚N, 145.00–168.75˚W) from
October 8 to 27, 2019. Zooplankton samples were collected using
vertical tows and a ring net (mouth diameter: 80 cm, mesh size: 335µm) and a quad-NORPAC (North Paciﬁc) net (mouth diameter: 45
cm, mesh sizes: 150 µm and 63 µm; Hama et al., 2019) at 10 m above
the bottom or 150 m to the surface at 40 stations (Figure 1;
Supplementary Table 1). The volume of the water ﬁltered throughthe NORPAC net with the 150-µm mesh was estimated using a oneway ﬂow meter (Rigosha CO., Ltd., Bunkyo-ku, Tokyo, Japan)
mounted in the mouth of the net for quantitative collection of all
copepodite stages in C. glacialis. Temperature, salinity, dissolved

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FIGURE 1

Location of sampling stations in the Paciﬁc sector of the Arctic Ocean. Solid circles indicate where NORPAC net (150-µm mesh) sampling of
Calanus glacialis/marshallae occurred in October 2019. The currents were adapted from Grebmeier et al. (2006).

The remaining sorted fresh samples were desalted using Milli-Q(Millipore Inc.), stored in 3-mL ointment bottles, and frozen at -80°
C until the fatty acid composition was analyzed.

oxygen, and turbidity were simultaneously measured using verticalcasts from a conductivity temperature depth (CTD) sensor (SeaBird Electronics Inc., SBE 911plus).
NORPAC net samples (mesh size: 150 µm) were immediately
preserved in 5% v/v borax-buffered formalin to analyze theabundance and population structure of C. glacialis. The
remaining samples collected by the ring net and the NORPAC
net (mesh size: 63 µm) were used to conduct gut pigment and fatty
acid composition analyses. Gut pigment analyses were conducted insamples from 11 stations on the Chukchi Plateau (77°N) obtained
during October 21–24, and fatty acid analyses were conducted insamples from throughout the study area.
With respect to the gut pigment analysis, 10% v/v soda
(saturated with CO2 in water) was immediately added to the fresh
samples after sample collection to avoid copepod grazing, gut
evacuation, and the decomposition of gut pigments (cf. Matsuno
et al., 2015). Fresh specimens of C. glacialis copepodite stage ﬁve(C5) were sorted using a stereomicroscope (M165C-Ergo/MCIHH/1; Leica Inc., Wetzlar, Germany). All specimens were sorted
under low-temperature and dim-light conditions within 1 h, andthe prosome length (PL) was measured using an eyepiecemicrometer attached to a stereomicroscope with an accuracy of
0.001 mm. The sorted specimens (two to ﬁve specimens, average:
three) were margined in small (2.5–3.3 mm) or large (3.4–3.8 mm)
categories and transferred into cuvette tubes ﬁlled with 6 mL of N,
N-dimethylformamide. One to four duplicates were obtained ateach station, resulting in a total of 31 and 28 gut piments samples
for the small and large categories, respectively. All samples were
kept under cool and dark conditions for at least 24 h to extract
chlorophyll and phaeopigments. After extraction, chlorophyll andphaeopigments were analyzed using a ﬂuorometer (10-AU-005;
Turner Designs Inc., California, United States), and the results werecombined and expressed as the digestive tract pigment content (µgpigment ind.-1; cf. Mackas and Bohrer, 1976).

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2.2 Sample analysis
In the laboratory, 19 zooplankton samples (Supplementary
Table 1) collected by the NORPAC net with a 150-µm mesh size
were split using a Motoda box splitter (Motoda, 1959). Calanus
glacialis was identiﬁed at the copepodid stage level based on
Brodsky (1967) in the aliquots and counted under a dissecting
microscope (SMZ1000, C-BD115, Nikon Corporation, Japan). To
calculate the total species abundance (ind. m-3), the count at eachstage was summarized and divided by the ﬁltered volume (m3) of
the net. Calanus marshallae is potentially distributed in thenorthern Bering Sea and Chukchi Sea (Frost, 1974), but many
zooplankton studies have treated this species as C. glacialis/
marshallae owing to difﬁculties in their identiﬁcation. Therefore,
as mentioned in the Introduction, we refer to specimens from the C.
glacialis/marshallae species complex as C. glacialis hereafter.
To determine geographical differences in the body size of C.
glacialis, the PL of at least 10 C. glacialis C5 individuals at eachstation was measured using an eyepiece micrometer attached to astereomicroscope with an accuracy of 0.001 mm. This measurement
was performed for all zooplankton samples (Figure 1).
The length of lipid accumulation relative to the PL (lipid
accumulation level) was measured at three levels: low, 0–4% of
PL; medium, 4–40% of PL; and high, >40% of PL (Matsuno et al.,2016). To compare C. glacialis data, the abundance (ind. m-2) was
calculated by multiplying the population density (ind. m-3) with thesampling depth to eliminate the inﬂuence of sampling depth
differences at each station. The mean C. glacialis copepodid stage(MCS) was calculated using Eq (1).

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6i  AiMCS = oi=16
oi=1 Ai

2.4 Data analysis(1)

To compare the biological parameters of C. glacialis C5 within
the Paciﬁc sector of the Arctic Ocean in 2019, a cluster analysis ofenvironmental data was conducted. In this respect, water column(from the surface to 10 m above the bottom or 150 m above thebottom) averages of hydrographic data (temperature, salinity,
dissolved oxygen, and turbidity) were calculated at each station.
The water column data were then standardized, and similarities
between stations were examined using the Euclidean distance. To
group stations, similarity indices were coupled with hierarchicalagglomerative clustering and the complete linkage method(unweighted pair group method with arithmetic mean, UPGMA)
using the SIMPROF test. Based on the dendrogram, stations wereseparated to deﬁne regions with similar hydrographic conditions. A
principal component analysis (PCA) was performed using
standardized hydrographic data to evaluate hydrographic
condition trends in each group. These analyses were conductedusing Primer 7 software (PRIMER-E Ltd.).
Non-metric multi-dimensional scaling (NMDS) ordination was
conducted using Primer 7 based on population structure, andPearson’s regressions of standardized hydrographic data werethen conducted to delineate the relationship between population
structure and hydrography. The abundance of each copepodid stage(ind. m-2) was transformed into that of the 4th root stage (X-4).
Similarities between samples were examined using the Bray-Curtis
index according to differences in the stage composition.
To obtain the fatty acid composition, we calculated sevenindices (docosahexaenoic acid (DHA)/eicosapentaenoic acid
(EPA), 16 polyunsaturated fatty acids (PUFA)/18PUFA, 18:2n-6,
15:0 + 17:0, PUFA/saturated fatty acid [SFA], 18:1n-9/18:1n-7, anddiatoms [D]/ﬂagellates [F]) for the prey record of C. glacialis(Kaneda, 1991; Budge and Parrish, 1998; Dalsgaard et al., 2003;
Stevens et al., 2004b; Alfaro et al., 2006).
Differences in biological parameters (abundance, MCS,PL,
MLS, and fatty acid composition) of C. glacialis C5 among
regions (identiﬁed by the cluster analysis based on hydrography)
were tested using the max-t method with a heteroscedastic
consistent covariance estimation (HC3) (Herberich et al., 2010).
The tests were conducted using R software with the packages“multcomp” and “sandwich” (version 4.1.2, R Core Development
Team, 2021). The relationship between the PL of C5 andtemperature was regressed using the Bêlehrá dek equation. A PCA
based on normalized seven-index data was performed for fatty acids
using Primer 7.

where i (1–6 indicates C1–C6) indicates the copepodid stage forC. glacialis, and Ai (ind. m-2) is the abundance of a copepodid stage(cf. Marin, 1987). Similarly, the mean lipid stage (MLS) of C.
glacialis C5 was calculated using Eq (2).
MLS = o3j=1 j  Bj

(2)

where j (1, 2, and 3 indicates low, medium, and high,respectively) indicates the lipid accumulation level, and Bj (ind.
m-2) is the ratio of the lipid accumulation level to the abundance.
Gonad maturation in adult females of the dominant copepod
species was classiﬁed as stage I (immature), stage II (small oocytes
in the ovary or oviduct), or stage III (large eggs or distended opacity
in the oviduct) (cf. Miller et al., 1984; Niehoff, 1998).

2.3 Fatty acid analysis
Fatty acid analyses were performed using a simpliﬁed direct
saponiﬁcation/methylation method (Matsumoto et al., 2018) afternecessary modiﬁcations were made for wax ester-containing
samples. Each C. glacialis sample was transferred to a preweighed screw-capped glass vial and dried in vacuo until aconstant weight was achieved. Methyl tricosanoate and 1tricosanol in toluene (2 mg mL-1 and 1 mg mL-1, respectively; 1µL) and 1 M KOH in 95% ethanol (50 µL) were added to the vial,
and after being crushed with a stainless-steel rod, the sample was
directly saponiﬁed overnight in the dark at 23°C. The reaction was
stopped by adding 3 M HCl in methanol (33 µL; Sigma-Aldrich),and the solvents and excess HCl were evaporated under a stream ofnitrogen and then in vacuo. The fatty acids released into the vial
were methylated in 3 M HCl in methanol (100 µL) at 80°C for 10
min, and the reagent was evaporated under a stream of nitrogen.
The fatty acid methyl esters were puriﬁed via column
chromatography using silica gel 60 (Merck) and hexane/diethyl
ether (95:5, v/v), and fatty alcohols were recovered from the same
column by elution with hexane/diethyl ether (50:50, v/v) and stored.
The puriﬁed fatty acid methyl esters were analyzed via gas
chromatography using a GC-4000 gas chromatograph (GLSciences, Tokyo, Japan) and an InertCap Pure-WAX column (30m × 0.32 mm i.d., 0.25 µm ﬁlm thickness; GL Sciences, Tokyo,
Japan). The column temperature was programmed to increase from
90°C to 170°C at 20°C min-1, then to 240°C at 4°C min-1, and to be
ﬁnally maintained at 240°C for 25 min. The injector and detector
temperatures were set at 240°C. The carrier gas was helium at alinear velocity of 29.3 cm s-1 at 170°C. Methyl esters dissolved inhexane were injected into the column in the splitless injectionmode, which was held for 1 min. A ﬂame ionization detector(Shimadzu Corporation, Japan) and Shimadzu C-R3A integrator(Shimadzu Corporation, Japan) were used for detection andconducting peak area measurements. Peaks were identiﬁed bycomparing their retention times with those of sardine oil fatty
acids. The fatty acid content was calculated from the peak area
ratios of methyl tricosanoate added to the initial C. glacialis samples.

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3 Results
3.1 Regions and water masses
The cluster analysis of biological parameters using hydrological
data identiﬁed four regions in the Paciﬁc sector of the Arctic Ocean(Figure 2A). As these regions were clearly divided geographically,
they were named (from south to north) the southern shelf, northern
shelf, slope, and basin (Figures 2A, C). The PCA results showed that04

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A

B

C

FIGURE 2(A) Results of Q-mode clustering based on hydrographic variables by Euclidean distance connected with group average mode in the Paciﬁc sectorof the Arctic Ocean in October 2019. Red lines indicate non-signiﬁcant station groupings, as tested by SIMPROF. Labels show sampling stations.
(B) Principal component analysis with base variables showing four groups based on the cluster analysis. (C) Geographical distribution of the groups
identiﬁed by the cluster analysis in the Paciﬁc sector of the Arctic Ocean during October 2019.

3.2 Geographical abundance and body sizeof C. glacialis

temperature, salinity, and turbidity were higher on the southern
shelf and lower in the basin (Figure 2B). Salinity was exceptionallyhigh on the slope, and the dissolved oxygen level was high on thenorthern shelf and basin (Figure 2B).
Danielson et al. (2020) deﬁned water masses based on
temperature and salinity as follows: warm Coastal Water (wCW),
warm Shelf Water (wSW), Ice Melt Water, cool Coastal Water(IMW cCW), cool Shelf Water (cSW), Anadyr Water (AnW),
Modiﬁed Winter Water (MWW), Winter Water (WW), andAtlantic water and bearing basin water (AtlW and BBW)
(Figure 3A). Within the 20 m depth on the southern shelf, wSW
was dominant on October 8 but cSW occupied this depth range on
October 27 (Figure 3B; Supplementary Table 1). A comparison of
the temperature and salinity on these two dates showed that salinity
was similar (32–32.49 vs. 31.99–32.36) but temperature was lower
on October 27 (decreased from 3.77–5.85°C to 2.36–2.84°C). In thenorthern shelf region, the water masses differed because the stations
and currents were widely distributed. AnW was observed below adepth of 25 m at St. 54, and AtlW and BBW were found below
depths of 100 m and 125 m, respectively, on the slope. In the basin, asimilar vertical distribution was observed among the stations; thesurface layer (0–40 m or 0–60 m) comprised IMW cCW, while
cSW, MWW, or WW existed at depths below these water masses.

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The abundance of C. glacialis ranged from 126 to 2,968 ind. m-2,
with higher and lower abundances on the slope and shelf, respectively(Supplementary Figure 1). A comparison of the abundance and stage
composition between the regions showed that the southern andnorthern shelves exhibited low abundance but had a highcomposition of C4 and C5 (Figure 4A). On the southern shelf, C5
was predominant in early October (St. 13 and 18), whereas adultfemales and males were predominant in late October (Sts. 61 and 70).
The highest abundance of C3–C5 was observed on the slope. In thebasin, the early stages (C1 or C2) occurred at 80% of stations (8/10),
and the highest proportion of early stages was 44% (St. FP3; Figure 4).
A few adult females were observed in each region, and almost all
specimens showed stage I gonadal maturation (data not shown).
NMDS based on the population structure showed relatively good
separation with clustering by hydrography (Figure 4B). Among thehydrographic variables, temperature and turbidity had signiﬁcantrelationships with population structure. Of the basin group, Sts. 30,
34, and 35 were located slightly further from the main distribution of
the basin community owing to the inﬂow of cSW at these stations(cf. Figure 3).

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A

B

FIGURE 3(A) T-S diagram in the Paciﬁc sector of the Arctic Ocean in October 2019. The colored symbols indicate the four groups identiﬁed via cluster
analysis (cf. Figure 2A): wCW, warm Coastal Water; wSW, warm Shelf Water; IMW cCW, Ice Melt Water and cool Coastal Water; cSW, cool Shelf
Water; AnW, Anadyr Water; MWW, Modiﬁed Winter Water; WW, Winter Water; AtlW&BBW, Atlantic Water & Bering Basin Water (cf. Danielson et al.,2020). (B) Vertical distribution of water masses at each station in the Paciﬁc sector of the Arctic Ocean in October 2019. The x-axis labels indicate
groups identiﬁed by cluster analysis (cf. Figure 2A).

3.3 Relationship between abundance, fatty
acid index, gut pigment andgeographical distribution

The body size (PL) of C. glacialis C5 ranged from 2.4–3.8 mm
throughout the sampling region (Figure 5). The median PL value ofeach region showed an increasing trend from the Chukchi Sea
toward the Arctic basin, in the order of the southern shelf, northern
shelf, slope, and basin (Figure 5). Body size was well described by
the Bêlehrá dek equation (PL=23594 (T+76.17)-2.05; Figure 6).

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A comparison of the biological parameters of C. glacialis among
regions showed signiﬁcant differences in the abundance of C5, PL of C5,

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A

B

FIGURE 4(A) Abundance and stage composition of Calanus glacialis/marshallae in the Paciﬁc sector of the Arctic Ocean in October 2019. The x-axis labels
indicate groups identiﬁed via cluster analysis (cf. Figure 2). (B) Non-metric multi-dimensional scaling (NMDS) ordination based on population
structure with Pearson’s regressions of hydrography. Symbols indicate clustering groups (cf. Figure 2A), and numbers with symbols are station IDs.
Red lines indicate correlations between each copepodite stage. T, temperature; S, salinity; Tur, turbidity; Oxy, dissolved oxygen. *: p< 0.05.

t test, the northern shelf had high 15:0 + 17:0, DHA/EPA, and 18:2n-6
values (Figure 8), and the other groups had relatively high 16PUFA/
18PUFA, D/F, and PUFA/SFA ratios. There was no signiﬁcantdifference between the three groups (excluding the northern shelf),
and the variabilities within the southern shelf and slope were within thebasin variability.
In the basin, small (2.5–3.3 mm in PL) and large (3.4–3.8 mm inPL) forms occurred simultaneously (Figure 9). The cut off criteria
between the forms is a length of 3.3 mm. Both small and large
specimens were found in all 11 samples during October 21–24. The
gut pigment showed a distinct diel pattern in the small forms, withhigh values during the night but low values during the daytime.
However, the large forms did not exhibit a diel grazing activity
pattern. The fatty acid indices were not signiﬁcantly different
between the small and large forms (data not shown).

total fatty acid content per individual, and fatty acid index (16PUFA/
18PUFA, PUFA/SFA, and D/F; Table 1). The abundance of C5 wassigniﬁcantly higher on the slope than on the other regions. (Figure 4;
Table 1). Signiﬁcant differences in the MCS were detected, and it
decreased from south to north (Figure 4; Table 1). There was no
signiﬁcant difference in MLS among regions; however, a minimum
value was observed on the southern shelf (Table 1; Supplementary
Figure 2). The total fatty acid content per individual in the basin was
signiﬁcantly higher than that on the southern shelf (Table 1). With
respect to the fatty acid index, 16PUFA/18PUFA, PUFA/SFA, and D/F
were signiﬁcantly lower on the northern shelf, but DHA/EPA, 18:2n-6,
and 15:0 + 17:0 were higher, although not signiﬁcantly (Figure 7;
Table 1). The results of the PCA based on the fatty acid indices showedthat the northern shelf was distant from the distribution of the othergroups (Figure 8). As shown in the regional comparison using the max-

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FIGURE 5

Size distribution of C5 Calanus. glacialis/marshallae in each clustering group (cf. Figure 2A) within the Paciﬁc sector of the Arctic Ocean. Dashedlines indicate the median of the prosome length of the species in each region.

4 Discussion

Similar results were detected in our study for the Chukchi Sea
and Arctic Basin.
In the pan-Arctic Ocean, the size range of the PL of species fromeach region has been well reported: 2.9–3.8 mm in Kandalaksha Bay
and the White Sea (Kosobokova, 1999), 2.55–3.55 mm near the
Svalbard Islands (Choquet et al., 2018), 2.8–4.0 mm in Disko Bay(Nielsen et al., 2014), 2.4–3.6 mm in Kongsfjorden (Gabrielsen
et al., 2012), and 2.4–3.7 mm in the Canadian Arctic and on the
Atlantic coasts (Parent et al., 2011). Compared to those reports

4.1 Geographical variation in body size
The body size of copepods varies with the environment(temperature and food concentration; Deevey, 1960; Viitasalo
et al., 1995; Kobari et al., 2003). In the Chukchi Sea in 2004,
2009, and 2012, the body size of C. glacialis C5 was found to be
negatively correlated with temperature (Ershova et al., 2015).

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FIGURE 6

Relationship between prosome length of C5 Calanus glacialis/marshallae and integrated mean temperature in the Paciﬁc sector of the Arctic Ocean.
The symbols and error bars indicate the mean and standard deviation, respectively. The relationship was expressed using Bê lehrá dek equations. The
groups were identiﬁed via cluster analysis (cf. Figure 2A).

TABLE 1 Regional comparison of biological parameters of Calanus glacialis/marshallae in different areas of the Paciﬁc sector of the Arctic Ocean
during October 2019.

GroupsParameters

Max-t test + HC3
South_shelf

North_shelf

Slope

Basin

Abundance of whole stages (ind.
m-2)

328 ± 128266 ± 100

2412 ±
556

619 ±
302

Abundance of C5 (ind. m-2)

294 ± 135

187 ± 99.0

590 ±
6.88

200 ±
127

North_shelf2

Basin2South_shelf2

Slope1

MCS (Mean copepodid stage)

5.05 ± 0.05

4.68 ± 0.13

4.06 ±
0.04

3.67 ±
0.83

Basin3Slope3

North_shelf2

South_shelf1

Prosome length of C5 (mm)2.91 ± 0.21

3.11 ± 0.15

3.24 ±
0.26

3.34 ±
0.23

South_shelf3

North_shelf2

Slope1

Basin1

MLS (Mean Lipid Stage)

2.07 ± 0.53

2.54 ± 0.03

2.42 ±
0.08

2.48 ±
0.27

Fatty acid (µg ind.-1)

138 ± 30.7

230 ± 97.3

233 ±
77.8

305 ±
149

Slope1,2

Basin1

DHA/EPAa

0.47 ± 0.12

0.92 ± 0.24

0.30 ±
0.02

0.33 ±
0.10

16PUFA/18PUFAb

0.65 ± 0.27

0.28 ± 0.02

0.89 ±
0.12

0.45 ±
0.23

South_shelf1,2

Slope1

18:2n-6c

0.89 ± 0.14

2.30 ± 1.02

0.89 ±
0.20

0.92 ±
0.20

NS

15:0 + 17:0d

1.50 ± 0.16

2.31 ± 0.35

1.66 ±
0.37

1.23 ±
0.20

NS

PUFA/SFAe

2.02 ± 0.29

0.92 ± 0.18

2.11 ±
0.53

1.92 ±
0.34

South_shelf1

Slope1

18:1n-9/18:1n-7f

6.90 ± 1.82

6.66 ± 0.25

4.77 ±
1.63

6.02 ±
1.57

D/Fg

1.98 ± 0.47

1.62 ± 0.23

3.06 ±
0.37

3.26 ±
0.95

Slope1,2

Basin1NSNSSouth_shelf2

North_shelf1,2

NSNorth_shelf2

North_shelf2

Basin1,2

Basin1

NS

North_shelf2

South_shelf2

Different superscript numbers in the Max-t test + HC3 column indicate signiﬁcant differences between regions. NS, not signiﬁcant.
a:Dinoﬂagellates/diatoms, carnivory (Budge and Parrish, 1998); b: Diatoms/ﬂagellates (Budge and Parrish, 1998; Alfaro et al., 2006); c: Terrestrial detritus or green algae (Dalsgaard et al., 2003); d:
Bacteria (Kaneda, 1991); e: Carnivory (Stevens et al., 2004a); f: Carnivory or omnivory (Stevens et al., 2004b); g: Diatoms (16 polyunsaturated fatty acids (PUFA), 16:1n-7 + 16:1n-9, 20:5n-3)/
ﬂagellates (22:6n-3, 18PUFA, 18:2n-6; Dalsgaard et al., 2003).

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FIGURE 7

Regional comparison of biomarker indices in fatty acids of C5 Calanus. glacialis/marshallae in the Paciﬁc sector of the Arctic Ocean in October
2019. The x-axis labels indicate groups identiﬁed via cluster analysis (cf. Figure 2A).

C. glacialis is (Frost, 1974), may decrease the mean body size in boththese regions.
Two C. glacialis haplotypes exist in the study area: Bering Sea andArctic Basin populations (Nelson et al., 2009). The Bering Sea
population extends from the Bering Strait to the northern Chukchi
Sea owing to the inﬂow of Paciﬁc Ocean water (Nelson et al., 2009;
Ashjian et al., 2021). The inﬂow of Paciﬁc water usually peaks from
August to October (Woodgate, 2018) and is mainly composed ofwSW (Danielson et al., 2020). As individuals in the Bering Sea
population experience warmer conditions than do those in thefrom other regions, the PL size range in our study (2.4–3.8 mm) was
one of the largest, and this was because the border of the samplingregion was situated in the Paciﬁc sector of the Arctic Ocean. The
body size of C5 in the Canada Basin in October 1997 was reported
as 3.38 ± 0.03 mm (Ashjian et al., 2003), which is similar to our
results (basin: 3.34 ± 0.23 mm; slope: 3.24 ± 0.26 mm; Beaufort Sea:
3.46 ± 0.20 mm). However, smaller body sizes were observed on thesouthern and northern shelves in our study (2.91 ± 0.21 mm, and3.11 ± 0.15 mm, respectively). It is considered that the cooccurrence of Calanus marshallae, which is slightly smaller thanFrontiers in Marine Science

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FIGURE 8

PCA based on biomarker indices in fatty acids of C5 Calanus glacialis/marshallae in the Paciﬁc sector of the Arctic Ocean in October 2019. The sizeof the circle indicates the fatty acid content per individual.

established in the Chukchi Sea because of their short ﬂushingtime (Ashjian et al., 2021).
Timing of phytoplankton blooms shifts from south to north in
the Paciﬁc Arctic Ocean (Arrigo and van Dijken, 2011), subsequent
reproduction of C. glacialis is expected to be associated with that.
However, interestingly, it was suggested that the earliest
reproduction period occurred in June in the basin region. Thiswas potentially caused by the longer residence time of C5 than thatof the other stages: the developmental time from nauplius three tocopepodite ﬁve is almost similar for each molting, but the period
from C5 to molt adults is longer (Peterson, 1986). Alternatively, it ispossible that another previous generation survived because of the
low metabolic rate at cold temperatures and the low grazingpressure by ﬁsh (Mundy et al., 2017; Skjoldal, 2022).

Arctic Basin population, it is considered that the Bering Sea
population would be smaller than would the Arctic Basin
population be. However, a haplotype analysis could not be
conducted in this study because ethanol samples were consumed
for another scientiﬁc work, and the difference in the PL between the
Bering Sea and Arctic Basin populations, therefore, remains unclear.

4.2 Reproduction and growth
Calanus glacialis is believed to undergo capital and income
breeding depending on the food conditions (Plourde et al., 2005;
Daase et al., 2013). Although our sampling was conducted inautumn, and our results do not, therefore, contain evidence ofreproduction, the timing of reproduction in spring was backcalculated using the developmental time and embryonic duration(Corkett et al., 1986) (see Supplementary Figure 3, Supplementary
Table 3). This type of back-calculation helps to remove thesampling time gap among regions (Kimura et al., 2022). The
estimation showed that almost all individuals had been spawnedin July and August.
In the Paciﬁc sector of the Arctic Ocean, peaks in
phytoplankton blooms have been observed during May in the
Chukchi Sea, and in June and July in the Siberian and Beaufort
Seas (Arrigo and van Dijken, 2011). In relation to this seasonality,
early stages (C1 and C2) dominate within the southern Chukchi Sea(Ashjian et al., 2021). However, there was a timing gap between thephytoplankton blooms (May–July) and our estimated reproduction
period (July–August), which could have been caused by the shorter
development time for the back-calculation of reproduction timing(Supplementary Table 3). The developmental time was assessedusing the temperature from our sampling period (October);however, they survived in colder conditions (Spring andSummer). Since warmer temperature induce shorter development
time (Corkett et al., 1986), therefore, the estimated development
time was shorter than the in-situ value. Another hypothesis is that
northward currents ﬂushed out individuals during May–July, andindividuals transported from the Bering Sea could not be

Frontiers in Marine Science4.3 Southern shelf
In early October, the entire water column was occupied by
wSW, but the surface layer (0–20 m) changed to cSW in late
October, possibly due to cooling by low air temperature, as othershelf water masses (e.g., AnW and wCW) were not observed.
Regarding the population structure, C5 was predominant in early
October, whereas adults were predominant in late October. Basedon these results, the population of C. glacialis appeared to grow,with some adults developing in late October. Incubation
experiments revealed that 50% of C. glacialis individuals develop
from C5 to adulthood in 20 days at 10°C (Peterson, 1986). In ourdevelopment time estimation based on Corkett et al. (1986), it
would take 10 days to undergo this process at 10°C and 20 days at 3°
C (Supplementary Figure 3); therefore, it was not surprising thatsome adults were observed in October. A high abundance of adultfemales was observed in the Arctic Basin in February, but only a few
such individuals were found throughout the year (Ashjian et al.,2003). During its life cycle, C. glacialis is capable of diapause fromC4 and C5 when it descends into the deep sea, and rich lipids are
stored in its body during autumn and winter (Lane et al., 2008;
Ershova et al., 2015; Pinchuk and Eisner, 2017). Because a sufﬁcient

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Kongsfjord in the Atlantic Arctic Ocean during autumn in 1997 (FalkPetersen et al., 2009), 0.15 in the Fram Strait during autumn in 2003
(Søreide et al., 2008), and 0.22 in Northwater during the autumn of1999 (Stevens et al., 2004a). It is evident that this species can change its
diet depending on the time and location (Banas et al., 2016), and theresults here show that the species on the northern shelf in the study
area are highly dependent on dinoﬂagellates.
Considering the energy ﬂow in lower ecosystems, eutrophic
conditions induce the dominance of diatoms because their growthis rapid from ingesting rich nutrients; however, oligotrophic
conditions stimulate microbial loop production (Pomeroy, 1974).
In the oligotrophic state, bacteria act as primary producers using
dissolved organic carbon, and several predator-prey interactions(bacteria vs. heterotrophic nanoﬂagellates, heterotrophic
nanoﬂagellates vs. small ciliates) occur, reaching their production
into heterotrophic dinoﬂagellates (Jakobsen and Hansen, 1997;
Levinsen and Nielsen, 2002). Therefore, the number of transfers
between trophic levels is much greater under oligotrophic conditions,
resulting in a low transfer efﬁciency. This suggests that the Calanus
glacialis can survive in a low-productivity ecosystem. In addition,although a high 18:2n-6 value indicates that the species grazes ongreen algae of a terrestrial origin (Dalsgaard et al., 2003), there havebeen no reports on the feeding on green algae by this species in theocean; therefore, further research is required.

depth (> 200 m) is required for the diapause of this species (Hirche,
1991; Ashjian et al., 2003), the shallow bottom depth(approximately 50 m; Nelson et al., 2009; Ershova et al., 2015)
and short residence time within the Chukchi Sea (Ashjian et al.,2021) imply that the population cannot be sustained/established.
The northward transportation of Paciﬁc zooplankton has beenreported in the Paciﬁc Arctic Ocean during warm years (Matsuno
et al., 2011; Ershova et al., 2015). Calanus marshallae, which was
originally distributed in the Northern California Current system tothe coastal Gulf of Alaska and the Bering Sea (Frost, 1974), should be
included within the Paciﬁc species; however, many studies have notdistinguished it from C. glacialis owing to the morphologicalsimilarities between the species. Genetic approaches have shownthat C. marshallae is present in the Chukchi Sea during the summer(Nelson et al., 2009 and Ashjian et al., 2021). In addition, thenorthward ﬂow at the Bering Strait is increasing (0.01 Sv per year),and residence time in the Chukchi Sea has decreased by ~1.5 months(from 7.5 months to ~ 5 months) based on mooring results from 1990
to 2019 (Woodgate and Peralta-Ferriz, 2021), which subsequentiallyextend C. marshallae distribution. Therefore, the inﬂow of C.
marshallae potentially distorts any changes identiﬁed in the C.
glacialis population, especially in the shelf region during warm
years. Genetic analyses are therefore required to avoid this distortion.

4.4 Northern shelf
4.5 Slope
The population structure of this area mainly comprises C4 andC5 C. glacialis. Assuming that this population on the shelf originatesfrom water masses similar to those on the southern shelf (wSW andcSW) that occupy more than half of the water column, thepopulation on the northern shelf (i.e., C4 and C5) is younger
than that on the southern shelf (C5). This may be attributed tothe shorter intermolt period at warmer temperatures (Corkett andMcLaren, 1978; Supplementary Table 3). The effect of temperature
on copepod growth has also been observed during warm and cold
years in the Chukchi Sea (Matsuno et al., 2011).
High variability in hydrography and water masses was observed inthis study, and the fatty acid composition of the specimens collected on
the northern shelf was lower in terms of 16 PUFA/18PUFA, PUFA/SFA, D, and F than that of specimens from other areas. The fatty acidcomposition is a valuable index for prey records because thecomposition of predators reﬂects that of their prey (El-Sabaawi et al.,2009). This result implies that individuals on the northern shelf do not
actively utilize diatoms or protozoans (Stevens et al., 2004a; Stevens
et al., 2004b). In contrast, higher values of DHA/EPA, 18:2n-6, and15:0 + 17:0 were observed in specimens on the northern shelf than inthose from other areas. Docosahexaenoic acid is abundant in
dinoﬂagellates, and EPA is abundant in diatoms (Budge and Parrish,1998), and the high DHA/EPA ratio, therefore, suggests that thisspecies grazes mostly on dinoﬂagellates within this area. However, thisfeature is considered to be rare in the Arctic Ocean compared with thatin the well-studied Atlantic Arctic Ocean. A survey in the southern
shelf area west of Spitsbergen in July 2018 showed a DHA/EPA of 0.67
(Trudnowska et al., 2020), which is lower than that observed in ourstudy (0.92). In addition, a DHA/EPA value of 0.21 was observed in

Frontiers in Marine Science

The highest abundance was observed on the slope, and theseresults are consistent with those obtained during studies conductedin August (Lane et al., 2008; Lliná s et al., 2009) and September(Ershova et al., 2021). In this area, easterly winds stimulate upwelling
along the shelf-break (Pickart et al., 2013), resulting in the occasionaltransport of diapause individuals (mainly C4 and C5) from the deep
sea to the surface (Lane et al., 2008; Lliná s et al., 2009). AtlW andBBW were only observed in this region below depths of 100 m or 125
m, which suggests the presence of upwelling (Carmack and Kulikov,
1998). A higher abundance was therefore observed on the slope
because of the presence of individuals derived from the deep sea via
upwelling (Pickart et al., 2013; Rutzen and Hopcroft, 2018). Anothermechanism that increases abundance is anti-cyclonic, cold-core eddy
formation from the boundary current ﬂowing along the Chukchi
Shelf (Pickart et al., 2005; Lliná s et al., 2009). Although theaccumulation of zooplankton by eddies is not very strong (Lliná s
et al., 2009), the formation rate of eddies is very high, with up to 1–2
eddies per day from early spring to early autumn (Pickart et al., 2005).
As previously mentioned, although shelf populations are ﬂushed out
into the basin (Ashjian et al., 2021), a high abundance is frequently
observed in this region, and this could potentially be related to theincreased eddy formation rate.
The population structure derived in our study is considered toreﬂect population growth, and this has also been determined in otherstudies. In August, early stages (C1 and C2) are abundant (Lane et al.,2008), C1–C4 are dominant in September (Ershova et al., 2021), andC3–C5 are dominant in October (this study). The abundance alters
between months, and it decreases from August (mean 18,910 ind. m-

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A

B

FIGURE 9(A) Lateral view of small and large specimens taken at St. MIZ-07 (cf. Supplementary Table 1). The three on the right-hand side were small with richgut pigments; the three specimens on the left-hand side were large with minimal gut pigment. (B) Comparison between grazing activity of small andlarge specimens in the basin region (cf. Figures 1, 2A) during October 21–24, 2023 (Supplementary Table 1). The local time is Alaska Standard Time
(UTC: 9 h). The area of the circle represents the amount of gut pigment (µg pigments ind.-1). The gray and white backgrounds represent nighttime
and daytime, respectively. The prosome length was determined from the mean value of two-ﬁve specimens used in the gut pigment analysis (cf.
Methods and Materials).

2, Lliná s et al., 2009) and September (mean 8,000 ind. m-2, Ershova
et al., 2021) to October (1,855-2,968 ind. m-2). It is of note that all
the abundance data were based on the samples collected using the150 µm mesh. Assuming the mortality rate for broadcast spawner
based on ﬁeld measurements (Hirst and Kiørboe, 2002), a valueof 0.0335 d-1 was calculated at 0.27°C (Supplementary Table 3).
Using this rate, the abundance in October was expected to be 2,448
ind. m-2 (=18,910 ind. m-2×(1-0.035)^(60 days)) from August and2,878 ind. m -2 (=8,000 ind. m-2×(1-0.035)^(30 days)) from
September. Predation mortality is independent of ambient
temperature, and the ratio of predation mortality to total mortality
increases to 3:4 in colder situations (Hirst and Kiørboe, 2002). This
indicates that the decrease in abundance on the slope was mainly
caused by predator feeding. A large biomass of polar cod has been
observed in Atlantic Water along the slope (Crawford et al., 2012).
Therefore, the high biomass of C. glacialis is due to physicalprocesses, and the rich prey is grazed on by organisms in thehigher trophic levels, such as ﬁsh, birds, and marine mammals(Dickson and Gilchrist, 2002; Bengtson et al., 2005; Crawford
et al., 2012).

Frontiers in Marine Science

With respect to the fatty acid composition, this region had thelowest DHA/EPA ratio and the highest 16PUFA/18PUFA ratio
among the four regions, which indicates that individuals in this area
consume more diatoms than do dinoﬂagellates and ﬂagellates. This
area is prone to upwelling (Pickart et al., 2013), which supplies richnutrients from deeper layers to the euphotic layer (Pickart et al.,2013; Rutzen and Hopcroft, 2018). Consequently, silicate
concentrations in the northern Chukchi Sea increase, providing asuitable environment for diatom growth (Hill and Cota, 2005;
Nishino et al., 2015). Therefore, geographical and physicalfeatures (i.e., upwelling) affect the C. glacialis population, andlipid accumulation relates to diatom grazing on the slope.

4.6 Basin
Based on the environmental data, Sts. 30, 34, and 35 werelocated in the basin, and the population structures at these stations
differed considerably from those of the other stations on the
Chukchi Plateau. This may be due to the transportation of shelf

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10.3389/fmars.2023.1168015(Choquet et al., 2018), but this result was impossible to clarify
because of a lack of genetic analysis.
An inter-form difference in the diel patterns of grazing activity
was observed. Small forms show higher gut pigment levels at night
than during the day, which is associated with diel vertical migration(DVM) (Conover and Huntley, 1991). The magnitude of DVM for
Calanus spp. varies with season and copepodid stage, and the DVM
intensity is greater during spring and autumn, when the dielchanges in light penetration are large (Falkenhaug et al., 1997).
During spring, C. glacialis responds sensibly to phytoplankton
dynamics via DVM, reaching sea ice and exhibiting diel patterns
in grazing activity (Runge and Ingram, 1991). In September, in thenorthern Chukchi Sea, diel changes in gut pigments have beenreported during small autumn phytoplankton blooms (Matsuno
et al., 2015). In contrast, DVM and active grazing ceases during thenight for Calanus spp., as determined by the lipids accumulated in
C5 (Falk-Petersen et al., 2008; Matsuno et al., 2015). Based on thisknowledge, it is hypothesized that the small forms perform DVM
and graze at night, while the large forms cease DVM and active
grazing and stay in the deep layer. Although our sampling design
could not corroborate this hypothesis, both forms were distributed
within depths of 0–150 m due to their co-occurrence during the day
and night (Falk-Petersen et al., 2008).

individuals by cSW inﬂow to depths of 50–100 m (Lane et al., 2008;
Lliná s et al., 2009). Previous studies found that the C5 stage
dominates the basin population in autumn (Ashjian et al., 2003;
Hopcroft et al., 2005). However, in our study, C5 was not abundant.
The early copepodite stages (C1 or C2) occurred at 80% of the basin
stations, and the maximum population percentage composition was
44%. Similar results, such as the occurrence of early stages on thesurface of the basin, have been reported in late summer (Hopcroft
et al., 2005) and winter (Ashjian et al., 2003). However, thecomposition of the early stage was lower than 10% because thisspecies is capable of diapause after C4 (Falk-Petersen et al., 2009;
Daase et al., 2013), and it is likely that C1 and C2 cannot overwinterin the basin because of their short growth periods (Ji et al., 2012).
With respect to the population origin of the early stages, adultfemales were observed at some stations in the basin, but all
specimens were immature. However, according to thedevelopmental time (Supplementary Table 3), the early stages
were spawned in August, and C5 was spawned in late June. Thisshows that long-term reproduction is supported by mixed
reproductive strategies (Plourde et al., 2005; Daase et al., 2013).
Primary production on the Chukchi Plateau is very low (Codispoti
et al., 2013), but the region is greatly affected by the inﬂow of Paciﬁc
Summer Water (PSW), which transports heat and organisms via
eddies (Watanabe et al., 2014; Muramatsu et al., 2021). The
composition of PSW in the basin has increased in recent years
compared to that during the 1990s and the 2000s (Bourgain andGascard, 2012; Muramatsu et al., 2021). Therefore, more organisms
are transported by PSW with eddies (Watanabe et al., 2014), andthis extends the reproduction period of the species. Potentially due
to the extended reproduction period, the early stages eventually
become more abundant than those in the results of Ashjian et al.
(2003) and Hopcroft et al. (2005). Field measurements of longevity
have shown that adults are quickly removed 16 days after
reproduction (Hirst and Kiørboe, 2002). These assumptions
suggest that adult females reproduce over the long term usingtransported organisms (e.g., phytoplankton), then that are removed
after reproduction. Similarly, overwintering individuals reproduce
in the Chukchi Sea following sea ice retreat, and the early stages aretransported to the basin (Ashjian et al., 2021). However, no genetic
evidence is available to support this hypothesis, and a haplotype
analysis of the early stages is required to reveal the extended
distribution and establishment of the Bering Sea population in
the basin (Ershova et al., 2021; Skjoldal, 2022),
Small and large forms co-occur on the Chukchi Plateau. On the
Atlantic side of the Arctic Ocean, it is difﬁcult to distinguishbetween C. glacialis and the smaller Calanus ﬁnmarchicus(Grainger, 1961). Choquet et al. (2018) investigated the possibilityof identifying species based on PL, morphological characteristics(the 5th pair of swimming legs and mandible), antennules, genital
somite pigmentation in live specimens, and genetic analyses. The
represented PL helps to approximate the species composition, but
no criteria for the species have been established; therefore, their size
ranges overlap (Choquet et al., 2018). In our case, the small
forms (2.5–3.3 mm) in C5 could be included as C. ﬁnmarchicus

Frontiers in Marine Science

5 Conclusions
This study describes geographic variations in the population
structure, body size, fatty acid composition, and diel changes in thegrazing activity of C. glacialis/marshallae in the Paciﬁc region of the
Arctic Ocean during autumn. The body size of this species wasfound to be negatively correlated with the water temperature fromthe Chukchi Sea to the Arctic basin. The population developed fromC5 was dominant on the southern shelf, and it was mostlytransported from the Bering Sea. C4 and C5 were dominant on
the northern shelf, suggesting that their growth was slower than thegrowth of those on the southern shelf. The fatty acid composition
revealed that the rate of prey dependence on dinoﬂagellates was
exceptionally high in the Arctic Ocean, indicating that the species
existed in a low-productivity ecosystem. The highest abundance andcomposition of diatom-derived EPA among the fatty acids were
found on the slope. This could be explained by the upwelling
accompanying the inﬂow of this species from the deep sea, andthe nutrient supply for the growth of diatoms. In the basin, earlystage individuals were more abundant than those reported inprevious studies because a larger number of organisms had
ﬂowed into the region, resulting in extended reproduction. Smalland large C5 forms were found, showing the different diel patternsof grazing activity. It appears that C. glacialis/marshallae haveadapted to the ﬂexibility in their life cycle caused by changes in
the physical and chemical environments of the Paciﬁc Arctic Ocean.
However, vertically stratiﬁed sampling and haplotype analyses are
required to monitor their biological features and reveal
population origins.

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JP18K14506 (Early Career Scientists), and JPJSBP120238801(Bilateral program)).

Data availability statement
The original contributions presented in the study are includedin the article/Supplementary Material. Further inquiries can bedirected to the corresponding author.

Acknowledgments
We thank the captain, ofﬁcers, crew, and researchers onboard
the R/V Mirai, the Canadian Coast Guard RV Amundsen, and IB
S.W. Laurier for their outstanding efforts during ﬁeld sampling.

Ethics statement
The manuscript presents research on animals that do not
require ethical approval for their study.

Conﬂict of interest
The authors declare that the research was conducted in theabsence of any commercial or ﬁnancial relationships that could beconstrued as a potential conﬂict of interest.

Author contributions
KM and AY designed the study. KT, KM, and KS performed
ﬁeld sampling. MI, KT, and YA analyzed the samples. MI and KM
analyzed the data and MI and KM wrote the paper with
contributions from all authors.

Publisher’s noteAll claims expressed in this article are solely those of the authorsand do not necessarily represent those of their afﬁliatedorganizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

Funding
This work was funded by the Arctic Challenge for Sustainability(ArCS; Program Grant Number JPMXD1300000000) and Arctic
Challenge for Sustainability II (ArCS II; program grant number
JPMXD1420318865) projects. This study was also supported by theEnvironment Research and Technology Development Fund(JPMEERF20214002) of the Environmental Restoration andConservation Agency of Japan and partially supported by theJapan Society for the Promotion of Science (JSPS) KAKENHI(grant numbers JP22H00374 (A), JP21H02263 (B), JP20K20573(Pioneering), JP20H03054 (B), JP20J20410 (B), JP19H03037 (B),Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fmars.2023.1168015/
full#supplementary-material

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