DOI: 10.5061/dryad.s7h44j1f1
title: Contribution of combined stressors on abundance and gene expression dynamics of the copepod Temora longicornis in the North Sea

description: 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.
description: <strong><em>Material &
 Methods</em></strong> Zooplankton
 were sampled with research vessel Simon Stevin on 33 sampling missions in
 2018 till 2021 (starting on 20<sup>th</sup> of February 2018,
 ending on 22<sup>nd</sup> 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) <em>Temora longicornis</em> 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 <em>T.</em>
 <em>longicornis</em> 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
 (NO<sub>3</sub><sup>-</sup>), Nitrite
 (NO<sub>2</sub><sup>-</sup>), Phosphate
 (PO<sub>4</sub><sup>3−</sup>), and Silicate
 (SiO<sub>4</sub><sup>4-</sup>) 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 <em>a</em> and
 <em>β</em>-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 Na<sub>2</sub>SO<sub>4</sub> and
 concentrated to about 5 mL using a rotary evaporator, followed by
 concentration under N<sub>2</sub> 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<sup>−1</sup>.
 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<sub>ow</sub>) 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. <em>Marine Pollution
 Bulletin</em>, 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. <em>Geoscience Data
 Journal</em>, 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. <em>Marine pollution
 bulletin</em>, <em>193</em>.
 https://doi.org/10.1016/j.marpolbul.2023.115159
description: # Contribution of combined stressors on abundance and gene expression
 dynamics of the copepod Temora longicornis in the North Sea
 [https://doi.org/10.5061/dryad.s7h44j1f1](https://doi.org/10.5061/dryad.s7h44j1f1) 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*. ## Description of the data and file structure The uploaded datafile gives a summary of plankton/environmental sample collections taken in 2018-2021, with sampling date and location and measured environmental parameters. Average copepod body length, prosome length and weight (body mass) are reported as well. It consists of one matrix: each column contains metadata (location and time of sampling, as well as the measured environmental variables, while each row represents a single, unique sample. Following parameters (in unique columns) can be found in the datafile: | Column title | Unit for parameter | Explanation parameter | | :------------- | :----------------- | :------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Date | - | Time of sampling | | Month | - | Sampling month | | Year | - | Sampling year | | Station | - | Name of sampling location (the station) | | Latitude | - | Coordinate of sampling location; Center latitude in decimal degrees, WGS84. | | Longitude | - | Coordinate of sampling location; Center longitude in decimal degrees, WGS84. | | Temp. | °C | Water temperature at 3 m depth | | Salinity | PSU | Salinity at 3 m depth | | Cond. | µS/cm | Water conductivity at 3 m depth; Electrical conductivity standard deviation of the water body by CTD, expressed in degrees µS/cm. | | Density | kg/m³ | Water density at 3 m depth | | Press. | db | pressure at 3 m depth | | Sound Velocity | m/s | Sound velocity at 3 m depth; The velocity of sound through the water body, expressed in m/s. | | Fluor. | mg/m³ | Fluorescence of the water body, at 3 m depth | | OBS | NTU | Optical back scatter, a measure for turbidity | | SPM | mg/L | Concentration of suspended particulate matter | | Secchi  depth | cm | Secchi depth, Visibility in the water body by Secchi disk, expressed cm. | | Day length | min. | Day length at day of sampling | | \[NO3-] | µmol/L | Concentration dissolved nitrate ions | | \[NO2-] | µmol/L | Concentration dissolved nitrite ions | | \[PO43-] | µmol/L | Concentration dissolved phosphate ions | | \[SiO44-] | µmol/L | Concentration dissolved silicate ions | | β Carotene | µg/L | Concentration of beta, beta-carotene {beta-carotene CAS 7235-40-7} per unit volume of the water body \[particulate &gt;GF/F phase] by filtration, acetone extraction and high performance liquid chromatography (HPLC); | | Chlor. a | µg/L | Concentration of chlorophyll-a {chl-a CAS 479-61-8} per unit volume of the water body \[particulate &gt;GF/F phase] by filtration, acetone extraction and high performance liquid chromatography (HPLC); | | Sum PAHs | ng/L | Concentration summed PAHs in water | | Sum PCBs | ng/L | Concentration summed PCBs in water | | Temora density | #individuals/m³ | Counted number of copepod Temora longicornis individuals in entire zooplankton sample (sampled with WP2 net), corrected for depth of water column | | Body mass | mg | Average body mass of an individual copepod T. longicornis | | Total Biomass | mg/m3 | Total biomass (body mass of all copepodsof species T. longicornis in a sample), corrected for depth of water column | | Prosome length | µm | Average prosome length of a copepod of T. longicornis | | Total length | µm | Average total length of a copepod of T. longicornis | Abbreviations used: Temp. = Temperature; Cond. = conductivity; Press. = Pressure, Fluor. = Fluorescence, OBS = optical backscatter, SPM = Suspended particulate matter. Min. = minutes. Chlor. = Chlorophyll. 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). The determination of suspended matter is done by filtration through a glass-fibre filter with a density between 50–100 g/m², and the amount of dried residue after dehydration, is measured. 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. 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 (SiO44-) were determined using a QuAAtro39 Continuous Segmented Flow Analyzer (SEAL Analytical GmbH, Norderstedt, Germany). 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. 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. 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). 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. For every sample, when possible, 30 animals of this species were picked out randomly and measured under a stereomicroscope (Olympus SZX2-ILLK; cellSens Dimension 1.13). Next to the copepod’s total body length (without antennae and furcal/caudal setae), we measured the length of the prosome.