DOI: 10.5061/dryad.x69p8czgs
title: In a comfort zone and beyond – ecological plasticity of key marine mediators

description: Copepods of the genus Calanus are the key components of zooplankton.
 Understanding their response to a changing climate is crucial to predict
 the functioning of future warmer high-latitude ecosystems. Although
 specific Calanus species are morphologically very similar, they have
 different life strategies and roles in ecosystems. In this study, C.
 finmarchicus and C. glacialis were thoroughly studied with regard to their
 plasticity in morphology and ecology both in their preferred original
 water mass (Atlantic vs. Arctic side of the Polar Front) and in suboptimal
 conditions (due to, e.g., temperature, turbidity, and competition in
 Hornsund fjord). Our observations show that ‘at the same place and time’,
 both species can reach different sizes, take on different pigmentation, be
 in different states of population development, utilize different
 reproductive vs. lipid accumulation strategies, and thrive on different
 foods. Size was proven to be a very mutable morphological trait,
 especially with regard to reduced length of C. glacialis. Both species
 exhibited pronounced red pigmentation when inhabiting their preferred
 water mass. In other domains, C. finmarchicus individuals tended to be
 paler than C. glacialis individuals. Gonad maturation and population
 development indicated mixed reproductive strategies, although a
 surprisingly similar population age structure of the two co-occurring
 species in the fjord was observed. Lipid accumulation was high and not
 species-specific, and its variability was due to diet differences of the
 populations. According to the stable isotope composition, both species had
 a more herbivorous diatom-based diet in their original water masses. While
 the diet of C. glacialis was rather consistent among the domains studied,
 C. finmarchicus exhibited much higher variability in its feeding history
 (based on lipid composition). Our results show that the plasticity of both
 Calanus species is indeed impressive and may be regulated differently,
 depending on whether they live in their ‘comfort zone’ or beyond it.
description: The study was performed in the Polar Front region in the southern
 part of the West Spitsbergen shelf in July 2018. It was designed to
 collect <i>Calanus </i>either from the water mass they
 originate from (<i>C. finmarchicus</i> from the Atlantic Water
 (AT station) domain, from the West Spitsbergen Current that carries warm
 and saline Atlantic Water and <i>C. glacialis</i> from the
 colder and fresher Arctic-type Sørkapp Current (AR station)), or from the
 Hornsund fjord, where two species co-occur, both in the main fjord basin
 (F station) and in the glacial bay (G station). The station representing
 the Atlantic water domain (AT) was characterized by the highest water
 temperature, salinity and chlorophyll fluorescence (Table 1). The lowest
 water temperature, salinity and chlorophyll fluorescence were observed at
 the station located in the glacial bay (G). However, the glacial bay was
 characterized by extremely high concentrations of particles.
 At each sampling station, two tows of a WP2 net (180 µm mesh
 size) were performed thorough the water column (70 m at G station, 200 m
 at F station, 170 m at AR station) or through the upper 200 m at the
 deep-water AT station. The sample from the first haul of the net was
 immediately fixed in a formaldehyde-borax solution for the analysis of
 <i>Calanus </i>abundance, developmental stage composition and
 gonad maturation stages. The samples from the next net hauls were put in
 buckets filled with seawater close to the <i>in situ</i>
 temperature. Then, the fifth copepodite stages of specific species
 (distinguished as small and large size modes) were photographed and
 preserved for further analyses. Overall, 1 252 photos
 of <i>Calanus</i> were taken and analyzed to determine the
 prosome length, the area of the lipid sac and the pigmentation. Methods
 for specific morpho-ecological traits were as follows: a. Size and species identification
 verification The
 prosome length of 1252 photographed <i>Calanus</i> CV
 copepodites was measured in ImageJ/Fiji free software for image analyses
 from the tip of the cephalosome to the distal lateral end of the last
 thoracic somite. The medians and quartiles of prosome length measurements
 are presented as box plots. Additionally, 991 copepodites were measured
 from the formaldehyde-fixed samples, among which 372 were CV copepodites,
 for which kernel density size distribution curves were fitted.
 Genetic species identification
 was used to test the accuracy of the applied morphological method of
 species discrimination for further analyses. Genetic identification was
 performed for each individual using its antennae and a set of six nuclear
 insertion–deletion markers (InDels) in a multiplex polymerase chain
 reaction (PCR) following the protocol described in Choquet et al. (2017).
 Accuracy of the morphological identification was calculated as the percent
 between the number of correct assessments in relation to the number of all
 assessments. Overall, the accuracy was 93%, but the accuracy was also
 calculated separately for each species and sampling location (Table 2).
 The species discrimination was  100% accurate at the fjord station (F) and
 almost fully accurate at the AR and AT stations. In the Atlantic domain,
 93% accuracy was obtained. In the Arctic domain, one individual with a
 size of 2.93 mm, initially identified as <i>C. glacialis,</i>
 turned out to be <i>C. finmarchicus</i>. A high percent of the
 species misidentification (22%) occurred only in the glacial bay G
 station. All the <i>C. glacialis</i> were properly identified,
 but many of the small copepodites, assumed to represent C.
 <i>finmarchicus,</i> turned out to be small <i>C.
 glacialis, </i>with individuals as small as 2.39
 mm<i>.</i> a. Red pigmentation
 The quantification of astaxanthin content was based on high
 performance  chromatography (HPLC) according to the methods described in
 Stoń-Egiert & Kosakowska 2005. Astaxanthin was isolated from
 previously lyophilized and weighed <i>Calanus</i> individuals
 by mechanical grinding in 90% acetone and sonication (2 min, 20 kHz, Cole
 Parmer, 4710 Series) for 2 h in darkness. Then, after clarification, the
 extract was subjected to chromatographic analysis. The HP1200  system
 (Agilent, Perlan Technologies) was equipped with a C18
 LichroCART™LiChrospher™ 100 RP18e (Merck) analytical column (dimensions
 250 x 4 mm, particle size 5 μm and pore size 100 Å). Pigments were
 separated in a gradient mixture of methanol, 1 M ammonium acetate and
 acetone. Calibration was conducted with commercially available standards
 (The International Agency for 14C Determination DHI Institute for Water
 and Environment in Denmark), which allowed for the qualitative assessment
  of astaxanthin (based on retention time and similarity with the
 absorbance spectrum of the standards) and  quantitative assessment  (based
 on response factor values obtained during the calibration
 procedure). Moreover, the pigmentation was analyzed by
 the visual examination of photos taken of live individuals by a color
 coding scheme, depending on whether there was a full (&gt; 50%, red),
 mid (10-50%, zebra), slight (&lt;10%, piece) or no (0%, transparent)
 coloration of antennae and prosome. The photos were also used to calculate
 the average pigmentation, where the rate of pigmentation was scored for
 each part of the body from 0 to 3 (e.g. antennae, prosome)  and 0 to 1
 (swimming legs, urosome) and summed, considering that approximately 10% of
 the pigmentation may be contributed by the urosome, 10% from the legs, 20%
 from the prosome and 60% from the antennas. b.
 Population development The relative abundances of each
 of the <i>Calanus </i>copepodite stages were used to describe
 the population age structure. The abbreviations (CI-AF) refer to six
 successive copepodite stages of <i>Calanus</i>, i.e., CI, CII,
 CIII, CIV and CV refer to the ﬁrst ﬁve copepodite stages, and AF to adult
 females. The species identification of particular developmental stages was
 based on the size discrimination assessed for the populations in the
 Hornsund fjord (Weydmann & Kwaśniewski, 2008). The age structure
 of specific species in the water domains studied was tested by Fisher’s
 exact test in R (fisher.test(data)), an independence test to determine if
 there is a significant relationship between two categorical
 variables. The gonad maturation stages of
 <i>Calanus </i>females were determined as described by Niehoff
 (2007) and Niehoff & Runge (2003). In our case, we distinguished
 stage G3, characterizing females that are preparing for spawning (multiple
 layers of oocytes in both anterior and posterior diverticula); stage G3.5,
 indicating females that are ready to spawn (ventral layer of oocytes is
 already colored, but not yet fully brown); and G4, representing females
 that would spawn within hours (oocytes undergoing final maturation in the
 most ventral layer in the gonads). The spent stage (S) characterizes
 females that had already finished reproduction. c.
 Lipid content Lipid content was determined as the mass
 of lipids that was extracted for the fatty acid composition analyses as
 well as estimated by the measurements of the lipid sac area. The lipid sac
 area was manually measured by contouring the sac perimeter by hand in all
 the photographed copepods. Furthermore, the specific equations derived
 from the reliable calibration of the individual lipid contents of Arctic
 copepods (Vogedes et al., 2010) were applied to calculate the total lipid
 content. Then, the percentage of the lipid sac area (fulfilment) was
 calculated as a function of the total area of the prosome. To verify if
 the lipid content differed between the two species, the unpaired
 two-sample Mann-Whitney test was performed, and to verify if the lipid
 content differed among study locations and species, the non-parametric
 Kruskal-Wallis test was performed in R (Package “stats”). The output of
 the Kruskal-Wallis test indicated if there is a significant difference
 between groups, but to know which pairs of groups are different, the
 function pairwise.wilcox.test was used to calculate pairwise comparisons
 between groups. d. Diet To verify
 the source of stored lipids in of the two species occurring at the same
 time in diverse water masses and in fjord waters, where they coexist, a
 combination of stable isotope (δ<sup>13</sup>C,
 δ<sup>15</sup>N) and fatty acid composition analyses were
 performed. Stable isotope analysis was performed
 following the protocol of Lebreton et al., (2012). Samples were analyzed
 using an elemental analyzer (Flash EA 1112, Thermo Scientiﬁc, Milan,
 Italy) coupled to an isotope ratio mass spectrometer (Delta V Advantage
 with a Conﬂo IV interface, Thermo Scientiﬁc, Bremen, Germany). The results
 are expressed in the δ unit notation as deviations from standards (Vienna
 Pee Dee Belemnite for δ13C and N2 in air for δ15N) following the formula:
 δ13C or δ15N = [(Rsample/Rstandard) - 1] x 103, where R is 13C/12C or
 15N/14N, respectively. Calibration was performed using reference materials
 (USGS-24, IAEA-CH6, IAEA-600, USGS-61, USGS-62 for carbon; IAEA-N2,
 IAEA-NO-3, IAEA-600, USGS-61, USGS-62 for nitrogen). Analytical precision
 based on the analyses of acetanilide (Thermo Scientiﬁc) used as laboratory
 internal standard was \0.1 and \0.15 ‰ for carbon and nitrogen,
 respectively. The identification and quantification of
 fatty acid methyl esters (FAMEs) was determined by gas chromatography/mass
 spectrometry (GC/MS) according to the method of Brown et al., 2011. An
 internal standard (10 μL; 1 mg mL<sup>–1</sup> nonadecanoic
 acid) was added to lyophilized and weighed <i>Calanus</i>
 samples. Samples were then saponified (20% KOH; 70°C; 60 min). Fatty acids
 were obtained by the addition of concentrated HCl (0.5 mL) to the
 saponified solutions followed by extraction into hexane (3x1 mL). Fatty
 acids were then methylated (1 mL; 1:9 HCl:MeOH; 80°C; 60 min) and
 re-extracted in hexane (3x1 mL) prior to analysis (Shimadzu QP2010 gas
 chromatograph coupled to a QP2020 quadrupole EI mass spectrometer; HP5ms).
 FAMEs were identified by comparison to authenticated standards (Supelco 37
 Component FAME Mix), retention times and mass spectral library matches
 (&gt;95% confidence). Instrumental abundances were normalized to the
 internal standard and sample mass for quantification.