Dynamics of Calanus Copepodite Structure during Little Auks’ Breeding Seasons in Two Different Svalbard Locations

Populations dynamics of key zooplankton species in the European Arctic, Calanus finmarchicus and Calanus glacialis (hereafter defined as Calanus) may be sensitive to climate changes, which in turn is of great importance for higher trophic levels. The aim of this study was to investigate the complete copepodite structure and dynamics of Calanus populations in terms of body size, phenology and their relative role in the zooplankton community over time in different hydrographic conditions (two fjords on the West Spitsbergen Shelf, cold Hornsund vs. warm Kongsfjorden), from the perspective of their planktivorous predator, the little auk. High-resolution zooplankton measurements (taken by nets and a laser optical plankton counter) were adapted to the timing of bird’s breeding in the 2015 and 2016 summer seasons, and to their maximal diving depth (≤50 m). In Hornsund, the share of the Calanus in zooplankton community was greater and the copepodite structure was progressively older over time, matching the little auks timing. The importance of Calanus was much lower in Kongsfjorden, as represented mainly by younger copepodites, presumably due to the Atlantic water advections, thus making this area a less favourable feeding ground. Our results highlight the need for further studies on the match/mismatch between Calanus and little auks, because the observed trend of altered age structure towards a domination of young copepodites and the body size reduction of Calanus associated with higher seawater temperatures may result in insufficient food availability for these seabirds in the future.


Introduction
Calanus finmarchicus and Calanus glacialis (hereafter Calanus) co-exist and dominate the mesozooplankton biomass in the shelf waters of the Arctic Ocean [1][2][3]. Originally, both species have different centers of distribution and thus are adapted to different environmental conditions and consequently adopt different life-history strategies [4][5][6][7]. As dynamic changes in the environmental conditions and in timing of primary production in the Arctic [8] cause high variability in the development rate and length of life cycles of the Calanus species [9], comprehensive phenological studies of these copepods are essential, especially in the context of their availability to planktivores in the warming Arctic. The important role of Calanus spp. in marine ecosystem functioning is based on the transfer of omega-3 fatty acids (long-chain PUFAs produced by marine algae), which are crucial for the growth and reproduction of all marine organisms [10]. Calanus spp. are full of lipids (up to 50-70% in dry mass) [11][12][13], and this makes them extremely nutritious for planktivores. Until recently, the amount of lipids was suggested to be species-specific, with a higher lipid content attributed to C. glacialis compared to the smaller C. finmarchicus [4,5,13]. This assumption emphasized the different role of these species in the Arctic ecosystem. However, in most of the studies the identification between these

Laboratory Analyses
Detailed laboratory analyses of each sample were performed according to a standard procedure, following the instructions given by Kwasniewski et al. [14]. First, zooplankters larger than 0.5 cm were removed from the sample, identified, and counted. Then, 2 mL subsamples were taken from each sample with a macropipette according to the sub-sampling method described by Harris et al. [54]. Subsamples were taken until at least 400 individuals were counted from a single sample. All organisms were identified to the lowest possible taxonomic level, typically species. Special focus was put on Calanus and their copepodite stages, which were identified according to criteria described by Kwasniewski et al. [55]. The abbreviations (CI-AF) refer to six successive copepodite stages of Calanus, i.e., CI, CII, CIII, CIV and CV as the first five copepodite stages, and AF to adult females. After

Sampling Protocol
Zooplankton samples from the little auk foraging grounds were collected during two consecutive summer seasons (hereafter study season) of 2015 and 2016 ( Figure 1 and Figure S1) in two study locations of cold Hornsund, and warm Kongsfjorden, both inside the fjords and in the adjacent areas located on the WSS (Figure 1). In Hornsund, samples were collected three times during each season, (hereafter study periods): three, 13, and seven samples in 2015 (hereafter respectively H1, H2, H3) and five, 15, and seven in 2016 (respectively H1 , H2 , H3 ). In Kongsfjorden nine, five, and six samples were collected in 2015 (hereafter respectively K1, K2, K3) and nine, eight, and nine in 2016 (K1 , K1.5 , K2 ). For exact sampling dates see Figure S1. In total, during 12 zooplankton sampling campaigns, 96 zooplankton samples were collected: 50 in the Hornsund area, and 46 in the Kongsfjorden area. Most sampling was performed directly from the R/V Oceania (IO PAN). Two sampling campaigns in the Kongsfjorden area (K1 and K2 ) were performed on board of the R/V Lance (Norwegian Polar Institute), one on board of a Zodiac boat (K3 in 2015) and one in Hornsund on board the S/Y Magnus Zaremba (H3 in 2016). Most of the samples (91) were collected using a WP2-type net (0.25 m 2 opening area) fitted with filtering gauze of 180 µm mesh size, which is best suited to catch all copepodite stages of Calanus. Five samples (K2) were collected using a WP2-type net with 60 µm mesh size. All zooplankton samples were collected from the mid-surface water layer (i.e., upper 50 m), which was arbitrarily chosen taking into consideration the little auk maximal diving depth of 50 m [53]. After collection, all samples were preserved in 4% formaldehyde solution in borax-buffered seawater and transported to laboratory for analysis.

Laboratory Analyses
Detailed laboratory analyses of each sample were performed according to a standard procedure, following the instructions given by Kwasniewski et al. [14]. First, zooplankters larger than 0.5 cm were removed from the sample, identified, and counted. Then, 2 mL subsamples were taken from each sample with a macropipette according to the sub-sampling method described by Harris et al. [54]. Subsamples were taken until at least 400 individuals were counted from a single sample. All organisms were identified to the lowest possible taxonomic level, typically species. Special focus was put on Calanus and their copepodite stages, which were identified according to criteria described by Kwasniewski et al. [55]. The abbreviations (CI-AF) refer to six successive copepodite stages of Calanus, i.e., CI, CII, CIII, CIV and CV as the first five copepodite stages, and AF to adult females. After microscope analysis, the number of individuals in each sample was converted into abundance (ind. m −3 ), basing on the volume of filtered water.
Due to the similar function of both species according to the trait-based approach [20] and difficulties in distinguishing them properly, for the purposes of this work C. glacialis and C. finmarchicus have been merged into one group, hereafter Calanus. However, the measurements of the prosome length of all copepodite stages of Calanus were performed for at least 30 individuals from each copepodite stage in the subsamples. To compare the prosome length of all the Calanus copepodite stages, 12,800 individuals were measured.

Temperature Measurements
Temperature data for comparison with the measurements of body size of Calanus and zooplankton community from net samples were obtained from 52 stations, (35 in Hornsund and 17 in Kongsfjorden) in two years of study by vertical profiles using a conductivity-temperature-depth sensor (CTD)). Measurements were first all binned into 1 m depth intervals, and then averaged over the upper 50 m water column for each station.

LOPC Measurements
To measure the size and concentrations of Calanus-type particles in different temperatures, the continuous oscillatory profiles, from the surface to 50 m depth, were performed with a conductivity-temperature-depth sensor (CTD; SBE 911plus, Seabird Electronics Inc., Washington, DC, USA) and a laser optical plankton counter (LOPC; Brooke Ocean Technology Ltd., Dartmouth, NS, Canada). The LOPC is an in-situ sensor that provides data on the abundance and size structure of a plankton community by measuring each particle passing through a sampling tunnel of a 49 cm 2 cross section. As the particle passes the sensor, the portion of blocked light is measured and recorded as a digital size and converted to the equivalent spherical diameter (ESD). The ESD is the diameter of a sphere that would represent the same cross-sectional area as the particle being measured with the use of a semi-empirical formula based on calibration with spheres of known diameters [56][57][58]. A Calanus-type group of particles, that involved only older life stages (app. CV), was selected basing on size (0.9-2.5 mm ESD) and transparency (attenuance index > 0.4) [59]. Then, the measurements (size + concentration of Calanus-type) were grouped according to a few seawater temperature ranges (<4 • C, 4-6 • C, and >6 • C) to calculate the 1d kernel density (R function geom_density) estimates of the dominating size in particular water temperatures.

Data Analyses
Multivariate nonparametric permutational ANOVA (PERMANOVA) [60] was used to test differences in: Calanus prosome length of individual copepodite stages; Calanus copepodite structure (CI-AF, stage index, and zooplankton species composition among fixed factors of regions and study periods. Prior to the analyses, abundance data were square-root-transformed [61]. The distribution of centroids representing particular samples was illustrated with a non-metric multi-dimensional scaling (nMDS) using Bray-Curtis similarities ordinations. The calculation of the Pseudo-F and p values was based on 999 permutations of the residuals under a reduced model [62]. To assess the magnitude of the spatial variation at each gradient, the estimated components of variation (ECV) as a percentage of the total variation were used. The relationship between seawater temperature and the Calanus prosome length and the mean size of zooplankton organisms was tested with the use of Pearson linear correlation. The Calanus stage index was calculated as a weighted mean on the basis of relative abundance of particular life stages, with each stage given values from 1 (CI) to 6 (AF) [63], where AF stage was represented by adult females. The mean size of zooplankton organisms in the sample was calculated as a weighted mean based on a local database with detailed measurements To determine zooplankton community structure, relative abundance of species/taxa was used. For each study period, a median number of individuals of a given taxa was calculated. Taxa constituting more than 5% of total zooplankton abundance were distinguished; the rest were grouped into an "others" category. To compare the prosome length frequency distribution of a copepodite stage CV of C. finmarchicus and C. glacialis, the number of all CVs in the sample (ind. m −3 ) was divided by the total number of measured individuals in the sample. To determine the relative abundance of measured individuals, each measurement of a given individual was multiplied by a factor: the abundance of CV (ind. m −3 )/number of measured individuals. Measured individuals were classified into size classes every 0.05 mm.

Size Response of Calanus and Mesozooplankton to Different Seawater Temperatures
The prosome length of Calanus copepodite stages based on comprehensive morphometrical analysis differed significantly between the two fjords in the case of all life stages (CI-CV), except for adults (AF) ( Table 1). In general, the median prosome length of Calanus individuals was larger in Hornsund than in Kongsfjorden ( Figure 2). The prosome length of individual copepodite stages of Calanus significantly correlated with temperature in both fjords for CI-CV (Pearson correlation coefficient, R = −0.37, p < 0.001 (CI); R = −0.48, p < 0.001 (CII); R = −0.39, p < 0.001 (CIII); R = −0.38, p < 0.001 (CIV); R = −0.37, p < 0.001 (CV), Figure 3), and did not correlate for AF (Pearson correlation coefficient, R = 0.01, p = 0.88, Figure 3). Generally, a relatively large variation in the body size of each Calanus copepodite stage was observed for each temperature recorded, except for the highest values (>7 • C), at which only relatively small individuals were observed in Kongsfjorden.    Additionally, a significant negative correlation between seawater temperature and mean zooplankton body size was observed (Pearson's correlation R = −0.46; p = 0.001; Figure S2). Larger animals (>1200 µm) were observed at lower seawater temperatures (i.e., below 4 • C) in Hornsund, while smaller organisms were dominating mainly in warmer (4-7.5 • C) Kongsfjorden.
Moreover, according to the measurements of the automatic LOPC method of the Calanus-type particles, individual size of older life stages of Calanus (CIV and CV) also differed in relation to the seawater temperature ( Figure 4). In Hornsund in both years the largest Calanus-type particles (c.a. 1.5 mm ESD) were observed in low temperatures (<4 • C), while the highest densities of smallest individuals (1.0-1.2 mm ESD) were noted at higher temperatures (>6 • C). In Kongsfjorden, the size range of older Calanus differed between two years, with larger size fraction dominating in 2015 ( Figure 4).

Zooplankton Community Structure
The zooplankton community structure in Kongsfjorden was significantly different between the two years ( Figure 5). In 2016, the Calanus percentage was relatively low, and decreased from 12% in K1′ and K1.5′ to 5% in K2′ ( Figure 5). The second numerous taxon, Oithona similis was more significant in 2015 (30% in K1 and 46% in K3) than in 2016 (20% throughout three study periods). The share of the Copepoda nauplii was higher (maximum in K1′) in 2016 than in 2015. Similarly as in Hornsund, the percentage of Bivalvia veligers were especially Calanus was the most abundant taxon in K1, when it reached 47% of the total zooplankton abundance. Its percentage decreased over time reaching 25% in K3 ( Figure 5). In 2016, the Calanus percentage was relatively low, and decreased from 12% in K1 and K1.5 to 5% in K2 ( Figure 5). The second numerous taxon, Oithona similis was more significant in 2015 (30% in K1 and 46% in K3) than in 2016 (20% throughout three study periods). The share of the Copepoda nauplii was higher (maximum in K1 ) in 2016 than in 2015. Similarly as in Hornsund, the percentage of Bivalvia veligers were especially high in 2016 (17-26%) and very low in 2015 (≤5% in K3). Another important zooplankton component in Kongsfjorden was Limacina helicina with 16% to 31% contribution to the overall zooplankton abundances in 2016. The percentage of Pseudocalanus spp. was rather constant over time. p = 0.003). In both years, Calanus dominated the zooplankton community, constituting approx. 60% of total zooplankton abundance in all three study periods of 2015 and approximately 40% in 2016. The second most numerous taxon was O. similis with at least 20% of total zooplankton abundance throughout all the periods of both study seasons ( Figure 5). Copepoda nauplii composed a significant percentage of total zooplankton abundance, especially in H1 and H1′ (20%). The contribution of Pseudocalanus spp. was in general the most important in H2 and H2′ study periods, while Bivalvia veligers comprised about 10% of all taxa in H2′ and H3′ in summer 2016.   The second most numerous taxon was O. similis with at least 20% of total zooplankton abundance throughout all the periods of both study seasons ( Figure 5). Copepoda nauplii composed a significant percentage of total zooplankton abundance, especially in H1 and H1 (20%). The contribution of Pseudocalanus spp. was in general the most important in H2 and H2 study periods, while Bivalvia veligers comprised about 10% of all taxa in H2 and H3 in summer 2016.

Calanus Copepodite Structure
The copepodite structure of Calanus in Kongsfjorden clearly differed between the years (PERMANOVA, MS = 6079.7, Pseudo-F = 15.19, p = 0.001) and between the study periods only in 2015 (PERMANOVA post hoc, MS = 1782.3, Pseudo-F = 4.45, p = 0.013). Calanus copepodite stage structures were much more similar among study periods within one year than among corresponding study periods of the two years ( Figure 6), which was indicated by higher estimated components of variance (ECV) for years other than for study periods (19. (PERMANOVA, MS = 3724.2, Pseudo-F = 31.16, p = 0.001), but also slightly between years (PERMANOVA, MS = 475.1, Pseudo-F = 3.98, p = 0.026; Figure 6). The higher share of ECV was obtained for the factor of periods than for a factor of year (15.9% vs. 4.4%). The Calanus copepodite stage index in Hornsund also differed between years (PERMANOVA, MS = 49.5, Pseudo-F = 7.62, p = 0.014) and study periods (PERMANOVA, MS = 746.9, Pseudo-F = 57.56, p = 0.001). Differences in the copepodite stage index had a stepwise character in both years, with a slightly higher median values in all investigated periods observed in 2016 (Figure 7). Large differences in Calanus copepodite structure in Kongsfjorden were only observed in 2015, when the proportion of early copepodite stages (CI-CIII) decreased from over 50% in K1 to about 10% in K3 (Figure 7). The CIV was the dominating life stage in K2, then it was outnumbered by CV in K3. The proportion of AF was very low with a slightly higher value, but not exceeding 2% in K1. In 2016, the composition of Calanus community was dominated by young copepodite stages and was very similar among study periods (Figure 7). The CI-CIII constituted approximately 60% of all copepodite stages with about 20% share for each stage. Calanus population in Hornsund in both years changed over time (Figure 7). Although the highest importance of the youngest copepodites of Calanus (CI and CII) were observed early in both years, (H1 and H1′), their contribution was almost two times higher in 2015 than in 2016. Thus, the correlation of these stages with the ordination coordinates was stronger concerning samples from 2015 ( Figure 6). The highest proportion of CIII was observed in 2015 in H2 (31%) and in 2016 in both H1′ and H2′ (25%). The highest proportion of CIV was observed in the second and the third study periods in both years and was slightly higher in 2016. Peak occurrence of CV took place in the third period of both years (H3 and H3′). The proportion of AF was relatively low throughout the study, but slightly higher in the first study periods of both years and correlated positively with the youngest life stages.

Discussion
The phenology of Calanus is a critical factor for little auks reproduction success during their breeding season, as was shown recently in a spatial perspective study in Greenland [44] and in time perspective study in Svalbard waters [64]. Therefore it is now of vital importance to study the match in time and space between the availability of older life stages of Calanus and little auks, because as was shown by this study, the development rate and the age structure of Calanus may differ significantly depending on the region, water temperature and time in the season. The issue is alarming not only because temperature warming has been shown to accelerate development of Large differences in Calanus copepodite structure in Kongsfjorden were only observed in 2015, when the proportion of early copepodite stages (CI-CIII) decreased from over 50% in K1 to about 10% in K3 (Figure 7). The CIV was the dominating life stage in K2, then it was outnumbered by CV in K3. The proportion of AF was very low with a slightly higher value, but not exceeding 2% in K1. In 2016, the composition of Calanus community was dominated by young copepodite stages and was very similar among study periods (Figure 7). The CI-CIII constituted approximately 60% of all copepodite stages with about 20% share for each stage. Calanus population in Hornsund in both years changed over time (Figure 7). Although the highest importance of the youngest copepodites of Calanus (CI and CII) were observed early in both years, (H1 and H1 ), their contribution was almost two times higher in 2015 than in 2016. Thus, the correlation of these stages with the ordination coordinates was stronger concerning samples from 2015 ( Figure 6). The highest proportion of CIII was observed in 2015 in H2 (31%) and in 2016 in both H1 and H2 (25%). The highest proportion of CIV was observed in the second and the third study periods in both years and was slightly higher in 2016. Peak occurrence of CV took place in the third period of both years (H3 and H3 ). The proportion of AF was relatively low throughout the study, but slightly higher in the first study periods of both years and correlated positively with the youngest life stages. The

Discussion
The phenology of Calanus is a critical factor for little auks reproduction success during their breeding season, as was shown recently in a spatial perspective study in Greenland [44] and in time perspective study in Svalbard waters [64]. Therefore it is now of vital importance to study the match in time and space between the availability of older life stages of Calanus and little auks, because as was shown by this study, the development rate and the age structure of Calanus may differ significantly depending on the region, water temperature and time in the season. The issue is alarming not only because temperature warming has been shown to accelerate development of Calanus [65], but also because the altered phenology of many species is becoming an increasingly important problem for trophic interactions [31,64,66] and thus entire food webs. To date, disturbance in interactions between predators and prey (match/mismatch) have been observed in many groups of organisms, e.g., between fish and plankton [67,68], insects and plants [69], birds and insects [70][71][72] shorebirds and arthropods [73] or seabirds and zooplankton [33,74,75]. The high variability in Calanus development, smaller body size and lower proportion in their concentration in relation to smaller zooplankton taxa in warmer Kongsfjorden, observed in our study, are in line with predicted scenarios of pelagic system modifications in the future Arctic towards faster development and prevailing role of smaller organism size [15,20,25]. Even though shortening life cycles and body size reduction of Calanus are expected not to have negative consequences for top predators [20], it will probably be important to the little auk, which is dependent on the availability of large, energy-rich prey [35].
Little auk preferentially search for large, lipid-rich copepods to cover the high energy costs incurred during foraging trips and feeding underwater [76][77][78]. Because the lipid content is strongly related to the body size, in this work Calanus size rather than species affiliation was utilized as the main qualitative trait. This approach is suggested as more appropriate for understanding the process of energy transfer to higher trophic levels on a larger scale [20]. Combining two species into one Calanus category was also supported by the recently discussed and clearly demonstrated problem of misidentification of C. glacialis and C. finmarchicus [17,19,20]. In order to gain a broader view on the Calanus population characteristics, in this study at the first step individuals of both species were identified and separated in accordance with traditional morphological classification [55], by measuring the prosome length. Results have shown that neither the specimens classified as C. glacialis nor C. finmarchicus were following a normal size distribution (Figure 8), which was in opposition to what was demonstrated for this stage for both species by molecular methods [20] (Figure 1). A right-skewed size distribution of C. glacialis both in Hornsund and in Kongsfjorden observed in our study is most probably caused by the fact that larger individuals classified as C. finmarchicus are in fact smaller individuals of C. glacialis [20]. This confirms that the size criterion is no more a reliable tool to accurately classify an individual for a given species [79], thus justifying our approach to combine them into one group.
Little auk preferentially search for large, lipid-rich copepods to cover the high energy costs incurred during foraging trips and feeding underwater [76][77][78]. Because the lipid content is strongly related to the body size, in this work Calanus size rather than species affiliation was utilized as the main qualitative trait. This approach is suggested as more appropriate for understanding the process of energy transfer to higher trophic levels on a larger scale [20]. Combining two species into one Calanus category was also supported by the recently discussed and clearly demonstrated problem of misidentification of C. glacialis and C. finmarchicus [17,19,20]. In order to gain a broader view on the Calanus population characteristics, in this study at the first step individuals of both species were identified and separated in accordance with traditional morphological classification [55], by measuring the prosome length. Results have shown that neither the specimens classified as C. glacialis nor C. finmarchicus were following a normal size distribution (Figure 8), which was in opposition to what was demonstrated for this stage for both species by molecular methods [20] (Figure 1). A rightskewed size distribution of C. glacialis both in Hornsund and in Kongsfjorden observed in our study is most probably caused by the fact that larger individuals classified as C. finmarchicus are in fact smaller individuals of C. glacialis [20]. This confirms that the size criterion is no more a reliable tool to accurately classify an individual for a given species [79], thus justifying our approach to combine them into one group. The body size of individual copepodite stages (CI-CV) of Calanus differed clearly between the two investigated fjords, with smaller prosome length observed in warmer Kongsfjorden than in colder Hornsund. First of all, such a difference may be explained by expected differences in proportions of Calanus species (C. glacialis vs. C. finmarchicus), which is also of importance for little auks [66]. But the change in individual size within particular species has also to be taken into consideration. Such observations are especially important in the light of recent studies demonstrating a great range of size plasticity of Calanus [17,19,20] and considering the fact that temperature is a key factor determining body size in copepods [21,80]. In this work, the smallest prosome length of Calanus CI-CV was observed at highest temperatures (˃7 °C), whereas the largest individuals were observed in seawater temperature ˂4 °C. A similar trend was observed by LOPC measurements of Calanustype particles selected for the older life stages, with the predominance of the largest individuals The body size of individual copepodite stages (CI-CV) of Calanus differed clearly between the two investigated fjords, with smaller prosome length observed in warmer Kongsfjorden than in colder Hornsund. First of all, such a difference may be explained by expected differences in proportions of Calanus species (C. glacialis vs. C. finmarchicus), which is also of importance for little auks [66]. But the change in individual size within particular species has also to be taken into consideration. Such observations are especially important in the light of recent studies demonstrating a great range of size plasticity of Calanus [17,19,20] and considering the fact that temperature is a key factor determining body size in copepods [21,80]. In this work, the smallest prosome length of Calanus CI-CV was observed at highest temperatures (>7 • C), whereas the largest individuals were observed in seawater temperature <4 • C. A similar trend was observed by LOPC measurements of Calanus-type particles selected for the older life stages, with the predominance of the largest individuals observed at temperatures below 4 • C and the smallest ones observed at temperatures above 6 • C in Hornsund. Such a clear differentiation in size modes can be explained by the co-occurrence of two water masses carrying two different Calanus species [14] and/or that the younger (CIV) copepodite stage was dominating in warmer, close to surface waters, while older (CV) life stage prevailed in colder conditions. Although the fraction of older Calanus-type particles was characterized by similar sizes in all temperature ranges in Kongsfjorden in 2015, the dominating size of this fraction was evidently shifted towards smaller sizes in the warmer season in 2016. This could be caused by different relative roles between CIV and CV life stages, or by the methodological bias, because the location of transects in both years slightly differed. In 2015 transects were performed closer to the interior part of the fjord, while in 2016 transects were mainly located in open shelf waters. Smaller individuals representing mainly C. finmarchicus can dominate in open waters with prevailing Atlantic water masses [19], while inside the fjord usually both species co-exist [17]. Hence the larger size of Calanus-type particles observed in 2015 could have resulted from a higher share of the larger species (C. glacialis). Despite differences in species composition, the significant relation between the body size of Calanus and seawater temperature observed in this study agrees with the assumptions of the temperature-size-rule (TSR) [81,82], which states that ectotherms grow slower, but mature at a larger body size in colder environments. The smaller size of Calanus in warmer temperatures observed in this study may be explained by the fact that organisms tend to be smaller in response to warming [83][84][85] and progressive reduction of Calanus body size is predicted with increasing seawater temperatures [86]. The body size of C. glacialis was found to vary considerably along its geographical range [19,20,83,87]. In experimental studies on C. finmarchicus, its prosome length also significantly decreased with increasing sea temperatures. Moreover, the largest individuals of C. glacialis were recorded after development in waters with temperatures not exceeding 3 • C [16].
In addition to individual size of Calanus, the proportion of Calanus in the overall community is very important for visual planktivores such as little auks, which need their prey to occur not only in a high concentration but also as easily visible [37]. In general, Calanus species are the key element of zooplankton communities in Svalbard waters, especially in terms of biomass [1,88,89], however their proportion in total zooplankton abundance is highly variable in time, space and under different hydrographic conditions [63]. In this study the proportion of Calanus in total zooplankton abundance was higher in Hornsund than in Kongsfjorden in both studied years, which confirmed more favourable foraging conditions for little auks in Hornsund than Kongsfjorden [77,90]. A similar predominance of Calanus in Hornsund was also recorded in 2007 [91]. Likewise, Trudnowska et al. [92] found higher proportions of Calanus in zooplankton communities in the colder Hornsund than in the Atlantic-influenced Magdalenefjorden. The lower proportion of Calanus in Kongsfjorden could result from the strong advection of the Atlantic Water carrying high concentrations of small copepods (e.g., Oithona similis) [27,93,94]. Therefore, the Atlantic water masses are typically avoided by little auks, due to high proportions of small copepods, which hinder detection of preferred prey [14,37]. The abundance of O. similis, which was really high in Kongsfjorden in this study, has been increasing gradually in Spitsbergen fjords since 2006 [91] as a consequence of the progressive Atlantification of these waters [27]. The increasing importance of small copepods in the zooplankton composition [89] is one of the most spectacular examples of the progressing warming that have already been documented [23].
Studies of Calanus development are challenging because its reproductive strategies are highly variable in time and space due to corresponding changes in environmental conditions and food supply [6,20,86,95]. In this study the development of the Calanus population and in consequence also its stage index, followed similar trends in Hornsund in both years. A similar gradual development of Calanus population, reflected by a dominance of young stages CI-CIII during the first study period and older CIV-CV stages during the third period was observed in Rijpfjorden [6]. Such similarity in the Calanus age structure observed in Rijpfjorden and Hornsund indicates a coincident timing of reproductive events and its synchrony with ice algae bloom in April and phytoplankton bloom in July [96]. In addition, the presence of early copepodite stages in all the studied periods (this study) might suggest continuous reproduction, or at least, the presence of more than one generation of Calanus, which is likely in high latitudes according to several new studies [20,65,97]. In Kongsfjorden, the trend of a gradual population development was observed in this work only in 2015. This observation coincided with the seasonal dynamic of the Calanus population structure emphasized for this fjord by a year-round investigation of Lischka and Hagen [94] with a higher contribution of early copepodite stages in July and a more advanced population in August. In turn in 2016, Calanus age structure was very similar in all three studied periods in Kongsfjorden and persisted relatively young according to low stage index. To some extent this might be caused by shorter time intervals between sampling periods in this year, but most probably it was caused by different advection impacts, according to different sea surface temperatures in the two years investigated (Figure 9). The events of advection are often associated with a transport of younger populations [55,93,95], which could explain the higher contribution of early stages in 2016 in Kongsfjorden in K1 (7-8 • C SST, Figure 9). This fact was also confirmed by a multiyear study conducted in the WSC region, where the copepodite structure of C. finmarchicus was younger during 'warmer' than 'colder' summers [27]. according to low stage index. To some extent this might be caused by shorter time intervals between sampling periods in this year, but most probably it was caused by different advection impacts, according to different sea surface temperatures in the two years investigated (Figure 9). The events of advection are often associated with a transport of younger populations [55,93,95], which could explain the higher contribution of early stages in 2016 in Kongsfjorden in K1′ (7-8 °C SST, Figure 9). This fact was also confirmed by a multiyear study conducted in the WSC region, where the copepodite structure of C. finmarchicus was younger during 'warmer' than 'colder' summers [27].

Conclusions
This study confirms the difficulty in proper Calanus finmarchicus and Calanus glacialis recognition, which, together with the similar functions of these species proved in recent studies, was an important argument for aggregating them into a single Calanus group. This study also provides evidence that the development rate and size structure of Calanus is highly variable in time, space and in relation to seawater temperature. Furthermore, according to the observations of this research, the copepodite structure is much more dynamically affected by Atlantification in Kongsfjorden than in Hornsund. Additionally, seawater temperature was confirmed to correlate negatively with both the mean size of mesozooplankton organisms and the body length of Calanus copepodites (CI-CV). These results support the hypothesis about shortening the life span and associated reduction of the body

Conclusions
This study confirms the difficulty in proper Calanus finmarchicus and Calanus glacialis recognition, which, together with the similar functions of these species proved in recent studies, was an important argument for aggregating them into a single Calanus group. This study also provides evidence that the development rate and size structure of Calanus is highly variable in time, space and in relation to seawater temperature. Furthermore, according to the observations of this research, the copepodite structure is much more dynamically affected by Atlantification in Kongsfjorden than in Hornsund. Additionally, seawater temperature was confirmed to correlate negatively with both the mean size of mesozooplankton organisms and the body length of Calanus copepodites (CI-CV). These results support the hypothesis about shortening the life span and associated reduction of the body size of Calanus along with climate warming. The accelerated development of Calanus can cause a significant shift in time in availability of its fifth copepodite stage in the foraging grounds of their key predator, the little auks. These findings confirm the hypothesis of the possible mismatch in timing between the availability of Calanus CV and the little auks highest food demands and therefore highlight the necessity to continue further seasonal studies of Calanus phenology in Svalbard waters.