The marine ecosystems of the Svalbard archipelago are now propelled towards a new climatic state owing to the ongoing “Atlantification” process. Fjords represent valuable natural laboratories hosting both local and advected populations of zooplankton. Our goal was to study life-history traits of two Calanus species (C. glacialis and C. finmarchicus) across slightly different environments in order to better understand their functioning within Arctic ecosystems undergoing substantial transformations. We hypothesized that life-history traits of Calanus copepods (CV life stage), such as size, pigmentation, lipid content, diet, parasite presence, and stage structure, would differ across four hydrographically distinct fjords (Hornsund, Isfjorden, Kongsfjorden, van Mijenfjorden) of Spitsbergen. Morphological size-based species identification via stereomicroscopy was supported by molecular methods. Manual image-based measurements of body size and lipid sack area were augmented by machine learning image analyses. Visual color intensity estimations were assisted by HPLC quantification of astaxanthin concentrations. Trophic variability was assessed via stable isotope analyses. The observed substantial variability in the life-history traits highlights their high plasticity and suggests that the traditional morphological distinctions between two Calanus species are becoming increasingly ambiguous. This underscores the need to incorporate genetic tools in ecological studies. The observed variability likely results from the coexistence of several cohorts of both species, including a mixture of local and advected populations. Moreover, each generation could be characterized by different traits depending on their source location and recruitment timing. These findings imply that under progressing “Atlantification” multiple adaptive responses may be expected, including reduced body size, accelerated development, mixed reproductive strategies, decreased pigmentation, shifts in diet, diversified lipid storage strategies, and increased parasite prevalence. Additionally, we introduce a machine learning-based tool for automatic assessment of key traits, such as body size and lipid content from images.

The study was performed in the summer of 2019 onboard RV Oceania in 4 fjords of the west Spitsbergen: Van Mijenfjorden (29^th of July), Hornsund (2^nd August), Isfjorden (5^th August), and Kongsfjorden (9^th August).

At each study location, two tows of a WP2 net (180 µm mesh size), proceded by a CTD profile, were performed through the water column (220 m in Hornsund, 215 in Isfjorden, 300 m in Kongsfjorden). The material collected from the first haul of the net was immediately fixed in a formaldehyde–borax solution as a sample for analysis via stereomicroscopy of Calanus abundance, developmental stage composition, gonad maturation stages, and size distribution. The material collected by the second net haul was used to select individuals only in the fifth copepodite stage of Calanus (CV), which were photographed alive as soon as possible after collection using a digital camera (Olympus SC50 CMOS Color Camera) mounted on an Olympus SZX16 stereomicroscope. The material was stored in a bucket in a refrigerator, and individuals were selected by subsampling to the counting chamber to guarantee their good condition for further analyses. Each individual was placed in a drop of water prior to making the high-resolution image (190 images were taken in H-Ar, 220 images in VM-iso, 257 images in I-AtAr, and 269 in the K-At region). The 922 images were used for the determination and measurements of prosome length, the area of the lipid sac, and pigmentation, and they were preserved for further analyses in the form of single individuals in alcohol for genetic analysis (220 individuals), pooled by 10 individuals per sample and frozen at -80°C for stable isotope (12 × 10 individuals) and HPLC analyses (41 × 10 individuals).

Genetic identification was performed for 216 Calanus CV individuals (50 from H-Ar, 50 from VM-iso, 60 from I-AtAr, and 60 from the K-At station). The analysis was based on six nuclear insertion–deletion (InDels) markers via a multiplex polymerase chain reaction (PCR) processing by following the protocol described in Choquet et al. (2017).

For the life stage structure analysis, 303 (H-Ar), 331 (ISF-ArAt), and 229 (K-AT) individuals were considered. In total, 890 individuals of Calanus were examined via these subsampling procedures. The relative abundances of each of the Calanus copepodite stages were used to describe the population stage structure. The abbreviations (CI-CV) refer to five successive copepodite stages of Calanus and AF refers to adult females. The stages in the gonad maturation stages of Calanus females included Gs1 and Gs2 as immature stages, Gs3 in the transition to maturity stage and Gs4 the final maturation stage (Niehoff & Runge 2003, Niehoff 2007).

The red carotenoid astaxanthin was detected and quantified via a chromatographic technique (HPLC). The pooled samples of 10 Calanus CV individuals were lyophilized and weighted before pigment extraction from their cells was conducted. The extraction procedure was based on mechanical grinding in the presence of 90 % acetone and sonication (2 min, 20 kHz, Cole Parmer, 4710 Series) for 2 h in the dark. The extract was subsequently clarified and injected onto a C18 LichroCART™LiChrospher™ 100 RP18e analytical column (250 × 4 mm dimensions, 5 μm particle size and 100 Å pore size, Merck). The qualification of astaxanthin was based on the retention time and similarity of the absorbance spectrum to the standard and its quantification on the basis of the response factor obtained during the calibration procedure (Stoń-Egiert & Kosakowska 2005).

The samples of pooled 10 individuals of Calanus CV for stable isotope analysis were analysed via an elemental analyzer (Flash EA 1112, Thermo Scientific, Milan, Italy) coupled with an isotope ratio mass spectrometer (Delta V Advantage with a ConFLo IV interface, Thermo Scientific, Bremen, Germany) according to standard protocols (Lebreton et al. 2012). 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] × 103, where R is 13C/12C or 15N/14N, respectively. Calibration was performed via reference materials (USGS-24, IAEA-CH6, IAEA-600, USGS-61, and USGS-62 for carbon; IAEA-N2, IAEA-NO-3, IAEA- 600, USGS-61, and USGS-62 for nitrogen). Analytical precision based on the analyses of acetanilide (Thermo Scientific) used as a laboratory internal standard was \0.1 and \0.15 ‰ for carbon and nitrogen, respectively.

The prosome length values of 922 photographed Calanus CV copepodites were measured from the tip of the prosome to the distal lateral end of the last thoracic somite. The measurements were performed with ImageJ/Fiji free software for image analysis.

The lipid sacs were manually measured by contouring the sac area by hand on photos of the Calanus CV. Fullness by lipids was calculated as a percentage of the lipid sac area within the total area of the prosome, whereas the amounts of wax esters were estimated by applying equations for image-based Arctic Calanus assessments (Vogedes et al. 2010).

References:

Choquet, M., M. Hatlebakk, A. K. S. Dhanasiri, et al. 2017. “Genetics Redraws Pelagic Biogeography of Calanus.” Biology Letters 13: 20170588.

Lebreton, B., P. Richard, R. Galois, et al. 2012. “Food Sources Used by Sediment Meiofauna in an Intertidal Zostera noltii Seagrass Bed: A Seasonal Stable Isotope Study.” Marine Biology 159: 1537–1550.

Niehoff, B. 2007. “Life History Strategies in Zooplankton Communities: The Significance of Female Gonad Morphology and Maturation Types for the Reproductive Biology of Marine Calanoid Copepods.” Progress in Oceanography 74: 1–47.

Niehoff, B., and J. A. Runge. 2003. “A Revised Methodology for Prediction of Egg Production Calanus finmarchicus From Preserved Samples.” Journal of Plankton Research 25: 1581–1587.

Stoń-Egiert, J., and A. Kosakowska. 2005. “RP-HPLC Determination of Phytoplankton Pigments—Comparison of Calibration Results for Two Columns.” Marine Biology 147: 251–260.

Vogedes, D., K. Eiane, A. S. B.tnes, and J. Berge. 2014. “Variability in Calanus spp. Abundance on Fine—To Mesoscales in an Arctic Fjord: Implications for Little Auk Feeding.” Marine Biology Research 10, no. 5: 437–448. https:// doi. org/ 10. 1080/ 17451 000. 2013. 815781.