The impact of multiple environmental and anthropogenic stressors on the marine environment remains poorly understood. Therefore, we studied the contribution of environmental variables to the densities and gene expression of the dominant zooplankton species in the Belgian part of the North Sea, the calanoid copepod Temora longicornis. We observed a reduced abundance of copepods, which were also smaller in size, in samples taken from nearshore locations when compared to those obtained from offshore stations. To assess the factors influencing the population dynamics of this species, we applied generalized additive models. These models allowed us to quantify the relative contribution of temperature, nutrient levels, salinity, turbidity, concentrations of photosynthetic pigments, as well as chemical pollutants such as polychlorinated biphenyls and polycyclic aromatic hydrocarbons, on copepod abundance. Temperature and Secchi depth, a proxy for turbidity, were the most important environmental variables predicting the abundances of T. longicornis, followed by summed PAH and chlorophyll concentrations. Analysing gene expression in field-collected adults, we observed significant variation in metabolic and stress-response genes. Temperature correlated significantly with genes involved in proteolytic activities, and encoding heat shock proteins. Yet, concentrations of anthropogenic chemicals did not induce significant differences in the gene expression of genes involved in the copepod’s fatty acid metabolism or well-known stress-related genes, such as glutathione transferases or cytochrome P450. Our study highlights the potential of gene expression biomonitoring and underscores the significance of a changing environment in future studies.

Material & Methods

Zooplankton were sampled with research vessel Simon Stevin on 33 sampling missions in 2018 till 2021 (starting on 20^th of February 2018, ending on 22^nd of December 2021) at four sampling locations in the Belgian part of the North Sea (BPNS). During 2018 and 2019, samples were collected in bi-monthly sampling campaigns. Due to the prevailing precautions prompted by the COVID-19 pandemic in 2020, the sampling strategy was adjusted for the remainder of the study period, leading to the collection of monthly samples due to the uncertain situation and prevailing regulations. For this study, two nearshore stations (130 and 230) and two offshore stations (215 and 330) were selected. Stations 130 and 230 are in proximity to the Ostend harbour in Belgium, with maximum depths of 13.8 and 16.7 m, respectively. Offshore station 215, maximum 29 m deep, is situated on the Flemish banks in the western region of the BPNS, while offshore station 330 (with a maximum depth of 25.9 m) is located more towards the eastern side, more in line with the nearshore stations. Stations 130, 230 and 330, situated in a near to offshore transect, were specifically selected as we hypothesized stronger fluctuations in environmental parameters in the more nearshore stations. In summary, 130 zooplankton samples (66 nearshore, 64 offshore) were collected.

Zooplankton were collected utilising a vertically towed WP2 net, which had a diameter of 57 cm and a mesh size of 200 µm. This net was equipped with a flow meter and was towed in an vertical manner from bottom to surface at each sampling station (max. speed of 1 m/s). From each sample, 25 adult (fully developed) and actively swimming (live) Temora longicornis specimens were picked out with a plastic transfer pipette in two replicates. These copepods were immediately stored in Invitrogen™ RNAlater™ Stabilization Solution, at a temperature of -20 °C, until they could be processed in the laboratory for RNA extraction. We chose to pool 25 individuals to 1) obtain sufficient RNA required for sequencing, and 2) be more representative of the population, decreasing the impact of individual variability. As handling the copepods affects specimen stress levels (and therefore may influence gene expression), we tried to minimise handling time as much as possible: it took approximately 5 to 10 min between the sample taking and storage of the copepods in RNAlater™. Due to time constraints and the requirement for specimens to be immobile for sex determination, sex ratio of the samples was not determined. The remainder of the zooplankton samples were preserved and kept in a 70 % ethanol solution, untill subsequent determination of T. longicornis density and morphometrics, using a Leica MZ 10 stereomicroscope.

At each sampling station, we conducted CTD (conductivity, temperature, and depth) profiling using a Seabird 25plusV2 CTD system (Flanders Marine Institute, 2021a). Additionally, the following environmental parameters were measured at a 3-meter depth in the water column: salinity (-), water density (kg/m³), sound velocity (m/s), optical backscatter (OBS; a measure for water column turbidity, expressed in Nephelometric Turbidity Units, NTU), Suspended Particulate Matter (SPM) concentration (mg/L), and Secchi Depth, a proxy for visibility (cm).

Water samples were obtained using Teflon-coated Niskin bottles at a 3-meter depth and later analysed for nutrient and pigment concentrations as part of the Flemish contribution to the LifeWatch ESFRI by Flanders Marine Institute (Flanders Marine Institute, 2021b). For nutrient analysis, 200 mL of seawater was filtered using cellulose-acetate filters to remove residual water, and the concentrations of Nitrate (NO₃^-), Nitrite (NO₂^-), Phosphate (PO₄³⁻), and Silicate (SiO₄^4-) were determined using a QuAAtro39 Continuous Segmented Flow Analyzer (SEAL Analytical GmbH, Norderstedt, Germany), as described in Mortelmans et al. (2019). For pigment analysis (chlorophyll a and β-carotene concentrations), seawater was filtered using a vacuum pump and Whatman GF/F glass fiber filters (47 mm) until the filter was saturated. After the filter ran dry, the sides of the sample container were flushed clean thoroughly with Milli-Q water. The filter was then stored in liquid nitrogen in a 2 mL storage unit. High-Pressure Liquid Chromatography (HPLC) was used to identify and quantify the pigments following the protocols outlined in Mortelmans et al. (2019).

Additionally, we quantified the concentrations of seven polychlorinated biphenyls (PCB 28, PCB 52, PCB 101, PCB 118, PCB 138, PCB 153, and PCB 180) and 15 polycyclic aromatic hydrocarbons (PAHs) at each sampling station. These analyses were performed on 5 L seawater samples, taken at a 3-meter depth, with 4 L retained for analysis. The concentrations of these toxicants were determined using gas chromatography-mass spectrometry (GC-MS), following liquid extraction of the filtered (0.7 µm) water samples. Internal standards (deuterated analogues of parent PAH compounds (Dr. Ehrenstorfer, VWR) and PCB congeners 14, 112, 143, 155 and 204 (Supelco/Dr. Ehrenstorfer, Sigma-Aldrich/VWR)) were added to the filtered 4L  samples, and were extracted three times with dichloromethane. The extracts were subsequently dried on Na₂SO₄ and concentrated to about 5 mL using a rotary evaporator, followed by concentration under N₂ to 0.2ml. Anthracene-d10 (Dr. Ehrenstorfer, VWR) was added as recovery standard. The extracts were concentrated and analysed with a gas chromatography–mass spectrometer (GC–MS, Thermoquest, Austin, Texas, USA). The extracts were injected (1 µL) into a 30 m × 0.25 mm DB5-ms cross-linked fused silica capillary column with a film thickness of 0.25 μm. Helium (99.999%) served as the carrier gas, at a linear flow of 1 mL min⁻¹. Splitless injection was performed at an injection temperature of 230 °C. Initially, the column was kept at 55 °C during injection, and then its temperature was ramped up at a rate of 15 °C per minute until it reached 310 °C, which was held for 6 minutes. The GC column was directly connected to the ion source of the mass spectrometer through a transfer line also heated to 310 °C. The quadruple MS operated in the selected ion monitoring electron ionization mode, with the ion source maintained at 250 °C. All solvents used were of purity suitable for organic residue analysis. As in Deschutter et al. (2017) and Semmouri et al. (2023), measured PAH/PCB concentrations were first multiplied with their corresponding octanol/water partition coefficient (K_ow) prior to conversion to molar concentrations.

References:

Deschutter, Y., Everaert, G., De Schamphelaere, K.A.C., & De Troch, M. (2017). Relative contribution of multiple stressors on copepod density and diversity dynamics in the Belgian part of the North Sea. Marine Pollution Bulletin, 125(1-2), 350–359. doi: 10.1016/j.marpolbul.2017.09.038

Mortelmans, J., Goossens, J., Amadei Martínez, L., Deneudt, K., Cattrijsse, A., & Hernandez, F. (2019). LifeWatch observatory data: Zooplankton observations in the Belgian part of the North Sea. Geoscience Data Journal, 6(2), 76-84. doi: 10.1002/gdj3

Semmouri, I., De Schamphelaere, K., Mortelmans, J., Mees, J., Asselman, J., & Janssen, C. (2023). Decadal decline of dominant copepod species in the North Sea is associated with ocean warming : importance of marine heatwaves. Marine pollution bulletin, 193. https://doi.org/10.1016/j.marpolbul.2023.115159