Contribution of combined stressors on density and gene expression dynamics of the copepod Temora longicornis in the North Sea.

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 density 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 generalised 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 (PAHs), on copepod density. Temperature and Secchi depth, a proxy for turbidity, were the most important environmental variables predicting the densities 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.

Decadal decline of dominant copepod species in the North Sea is associated with ocean warming: Importance of marine heatwaves.

Time-series are crucial to understand the status of zooplankton communities and to anticipate changes that might affect the entire food web. Long-term time series allow us to understand impacts of multiple environmental and anthropogenic stressors, such as chemical pollution and ocean warming, on the marine ecosystems. Here, a recent time series (2018-2022) of abundance data of four dominant calanoid and one harpacticoid copepod species from the Belgian Part of the North Sea was combined with previously collected (2009-2010, 2015-2016) datasets for the same study area. The time series reveals a significant decrease (up to two orders of magnitude) in calanoid copepod abundance (Temora longicornis, Acartia clausi, Centropages spp., Calanus helgolandicus), while this was not the case for the harpacticoid Euterpina acutifrons. We applied generalized additive models to quantify the relative contribution of temperature, nutrients, salinity, primary production, turbidity and pollution (anthropogenic chemicals, i.e., polychlorinated biphenyls and polycyclic aromatic hydrocarbons) to the population dynamics of these species. Temperature, turbidity and chlorophyll a concentrations were the only variables consistently showing a relative high contribution in all models predicting the abundances of the selected species. The observed heat waves which occurred during the summer periods of the investigated years coincided with population collapses (versus population densities in non-heatwave years) and are considered the most likely cause for the observed copepod abundance decreases. Moreover, the recorded water temperatures during these heatwaves correspond to the physiological thermal limit of some of the studied species. As far as we know, this is the first study to observe ocean warming and marine heat waves having such a dramatic impact (population collapse) on the dominant zooplankton species in shallow coastal areas.

Assessment of road run-off and domestic wastewater contribution to microplastic pollution in a densely populated area (Flanders, Belgium).

Plastics are omnipresent in our daily life. Unfortunately, the produced plastics will partly end up in the environment including aquatic ecosystems. People often refer to littering or illegal waste dumping as sources of plastic emission to the environment. However, daily-life sources could also, unknowingly, contribute considerably to the total microplastic pollution in the ecosystem. Hence, there is an urgent need to study these potential sources. In this research, two common sources, i.e. domestic wastewater and road run-off from tire and road wear particles, were studied in detail to quantify the relative contribution of both domestic sources towards microplastic pollution in freshwater ecosystems in Flanders, Belgium. This assessment shows that every person (in studied area) emits on average 1145 microplastics (25-1000 µm) daily through domestic wastewater, resulting in a yearly discharge of 418,000 microplastic particles per person. The road run-off samples contained between 0.02 and 9.2 mg tire wear particles per litre per day, which corresponds to an emission of 10.8 mg tire wear particles per driven vehicle km. The gross and net emissions of both above mentioned microplastic sources were extrapolated to the whole Flanders region using an emission model. From the yearly gross microplastic pollution in the domestic wastewater, 623 kg (20%) will be discharged in the freshwater. The highest losses originated from the households that have a private drain or are not (yet) connected to an active wastewater treatment plant. In Flanders, the yearly net microplastic emission into the aquatic environment of tire wear particles is estimated to be 246 tonnes (38%), mainly from the direct run-off from the road surface. Based on the results, specific mitigation measures can be installed to reduce the emission of microplastics towards the freshwater ecosystem. Other sources should be quantified in a similar way for a more holistic strategy to counteract plastic pollution.

Distribution of microplastics in freshwater systems in an urbanized region: A case study in Flanders (Belgium).

Microplastics (MPs) are an emerging pollutant of concern in all known aquatic ecosystems. However, studies at a regional scale on MP pollution in freshwater systems and the necessary risk assessments are limited. Therefore, in this study, we examined microplastic concentrations, size distributions, and polymer types in surface waters and sediments in the geographic region Flanders (Belgium), as a case study for a densely populated region and one of the most developed parts of Europe. Samples have been taken on nine different locations, of which five were repeated in a different weather condition. In total 43 aqueous and nine sediment samples have been collected. The quantity and identity of the microplastics in the samples were determined with µFTIR spectroscopy in the range of 25-1000 µm. The MPs' abundances in surface waters and sediments ranged from 0 to 4.8 MP L-1 (average = 0.48 MP L-1) and from 0 to 9558 MP kg-1 dry weight (average = 2774.57 ± 2317.93 MP kg-1 DW), respectively. Polystyrene and polypropylene were the most common polymer compositions found. No correlations were observed between microplastic concentrations in the sediment/the surface water samples and the measured environmental variables rainfall, conductivity, pH, dissolved oxygen content, waterway flow rate and width, and surrounding land use. Risk assessment results for the measured surface water concentrations through the risk quotient (RQ) method and the probabilistic risk assessment framework suggest that most of the sampled sites in Flanders posed negligible risks to freshwater biota, while this was not the case for some of the sediment concentrations. Our results illustrate the need to urgently develop analytical methods that can routinely measure the full size range of MP in environmental samples to adequately assess risks for the environment.

Toward a Better Understanding of the Contribution of Wastewater Treatment Plants to Microplastic Pollution in Receiving Waterways.

Microplastics (1 µm-5 mm), are ubiquitous in daily-use products and regularly end up in the wastewater. The main part of the wastewater is treated in wastewater treatment plants (WWTPs), which allow for at least partial removal of microplastics. The present study aimed to understand the contribution of domestic wastewater to microplastic pollution in Flanders (Belgium) via two main discharge routes of microplastics: (1) the effluent, and (2) removed fractions. Furthermore the effect of effluent discharge on the microplastic contamination in the waterway was studied in both surface water and sediment samples of upstream and downstream locations of the discharge from three WWTPs. On average, 12.64 ± 20.20 microplastic/L entered a WWTP (10 µm-5 mm). The effluent contained on average 0.41 ± 0.91 microplastic/L, resulting in an average removal efficiency of 97.46% ± 2.33%, which is comparable with various (non-)European countries. Removal efficiencies are both polymer- and size-specific, and data suggest that smaller particles are less efficiently removed from the wastewater, which also causes an increased input of smaller particles to the environment. The sludge is the most efficient treatment process to remove microplastics. Despite the high removal efficiencies, still 1.11 × 107 ± 3.07 × 107 microplastics end up in the nearby waterway daily. Nonetheless, based on the results gathered in the present study, this does not seem to impact the microplastic concentration in the waterway significantly. In summary, the present study offers a holistic approach in the research on the impact of wastewater on microplastic pollution in the ecosystem, integrating different discharge routes and measuring the impact on environmental microplastic pollution. Environ Toxicol Chem 2023;42:642-654. © 2022 SETAC.

Allometric scaling improves the characterization of complex community transcriptomes.

Allometric scaling, that is scaling patterns in physiological processes relative to body size, can be used to overcome the current limitations in metatranscriptomics. Metatranscriptomics refers to the use of RNA transcripts to characterize a complex community. In contrast to metagenomics, metatranscriptomics allows one to simultaneously address community composition and functionality through the characterization of the community transcriptome. Hence, insights into metabolic processes and molecular pathways can also be obtained. Despite its increasing use in community ecology, a major limitation and source of error is the variation in RNA transcript abundance across organisms varying in body size. Hence, this may lead to incorrect estimations of the community structure and functioning as larger RNA quantities from larger individuals may overestimate their community contribution.

Spatio-temporal patterns in the gene expression of the calanoid copepod Temora longicornis in the Belgian part of the North Sea.

Marine zooplankton are increasingly being affected by recent environmental changes, such as climate change, and respond with profound spatial relocations and shifts in phenology and physiology. In order to predict whether populations are able to persist or adapt to such new conditions, it is essential to understand the molecular basis of such adaptations, which ultimately get translated into these physiological responses. To explore variation in population gene expression across time and space, we investigated transcriptome-level profiles of the calanoid copepod Temora longicornis, that were collected at four different locations in the Belgian Part of the North Sea (BPNS) on three different time points (April, June, October) in 2018. RNA-seq analysis of field collected adults identified large seasonal differences in gene expression, mainly between spring-summer and autumn samples. The largest log-fold changes occurred in a set of genes encoding for ribosomal and myosin (heavy chain) transcripts. Enrichment analysis revealed a strong seasonal pattern in vitellogenin, cuticle and glycolytic gene expression as well. We also found a positive correlation between vitellogenin expression and densities of T. longicornis. No clear spatial variation in expression patterns was found in the BPNS. This study underlines the potential of field gene expression studies for biomonitoring purposes and the significance of considering seasonal variation in future studies.

Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community.

The implementation of cost-effective monitoring programs for zooplankton remains challenging due to the requirements of taxonomical expertise and the high costs of sampling and species identification. To reduce costs, molecular methods have been proposed as alternatives to morphology-based monitoring. Metatranscriptomics can contribute to promote both cost-effectiveness and accuracy of biological assessments of aquatic ecosystems. Here, we describe and evaluate the construction of a metatranscriptome dataset from a pelagic crustacean zooplankton community. We sampled zooplankton in one marine station, named LW02, in the North Sea, in both winter and summer, and generated transcripts using Oxford Nanopore Technology (ONT), a third-generation nanopore-based sequencing technology. ONT is, uniquely, capable of sequencing RNA directly, rather than depending on reverse transcription and PCR, and applicable to be used directly in the field. We found that metatranscriptomics is capable of species detection, including screening for the presence of endoparasites, hence competing with morphological identification. Taxonomic analysis based on ribosomal 18S transcripts identified calanoid copepods, particularly Temora longicornis and Acartia clausi, as the most abundant community members. Moreover, up to 40.4% and 50.5% of all sequences could be assigned to predicted genes in the winter and summer sample, respectively. The most abundant mRNA transcripts with known function coded for essential metabolic processes. GO term annotation revealed that genes involved in glycolytic and translation-related processes were most expressed in the community. Although small in scale, our study provides the basis for future efforts to characterize the metatranscriptome of marine zooplankton communities and its application in biomonitoring programs.

Genome-Wide Stress Responses to Copper and Arsenic in a Field Population of Daphnia.

Over the past decade, significant advances have been made to unravel molecular mechanisms of stress response in different ecotoxicological model species. Within this study, we focus on population level transcriptomic responses of a natural population of Daphnia magna Straus, (1820), to heavy metals. We aim to characterize the population level transcriptomic responses, which include standing genetic variation, and improve our understanding on how populations respond to environmental stress at a molecular level. We studied population level responses to two heavy metals, copper and arsenic, and their binary mixture across time. Transcriptomic patterns identified significantly regulated gene families and genes at the population level including cuticle proteins and resilins. Furthermore, some of these differentially regulated gene families, such as cuticle proteins, were also significantly enriched for genetic variations including SNPs and MNPs. In general, genetic variation was observed in specific gene families, many of which are known to be involved in stress response. Overall, our results indicate that molecular stress responses can be identified within natural populations and that linking molecular mechanisms with genetic variation at the population level could contribute significantly to adverse outcome frameworks.

The transcriptome of the marine calanoid copepod Temora longicornis under heat stress and recovery.

Understanding the impacts of global change in zooplankton communities is crucial, as alterations in the zooplankton communities can affect entire marine ecosystems. Despite the economic and ecological importance of the calanoid copepod Temora longicornis in the Belgian part of the North Sea, molecular data is still very limited for this species. Using HiSeq Illumina sequencing, we sequenced the whole transcriptome of T. longicornis, after being exposed to realistic temperatures of 14 and 17 °C. After both an acute (1 day) and a more sustained (5 days) thermal exposure to 17 °C, we investigated gene expression differences with animals exposed to 14 °C, which may be critical for the thermal acclimation and resilience of this copepod species. We also studied the possibility of a short term stress recovery of a heat shock. A total of 179,569 transcripts were yielded, of which 44,985 putative ORF transcripts were identified. These transcripts were subsequently annotated into roughly 22,000 genes based on known sequences using Gene Ontology (GO) and KEGG databases. Temora only showed a mild response to both the temperature and the duration of the exposure. We found that the expression of 27 transcripts varied significantly with an increase in temperature of 3 °C, of which eight transcripts were differentially expressed after acute exposure only. Gene set enrichment analysis revealed that, overall, T. longicornis was more impacted by a sustained thermal exposure, rather than an immediate (acute) exposure, with two times as many enriched GO terms in the sustained treatment. We also identified several general stress responses independent of exposure time, such as modified protein synthesis, energy mobilisation, cuticle and chaperone proteins. Finally, we highlighted candidate genes of a possible recovery from heat exposure, identifying similar terms as those enriched in the heat treatments, i.e. related to for example energy metabolism, cuticle genes and extracellular matrix. The data presented in this study provides the first transcriptome available for T. longicornis which can be used for future genomic studies.

Molecular phylogenetics of the genus Costularia (Schoeneae, Cyperaceae) reveals multiple distinct evolutionary lineages.

We investigated the monophyly of Costularia (25 species), a genus of tribe Schoeneae (Cyperaceae) that illustrates a remarkable distribution pattern from southeastern Africa, over Madagascar, the Mascarenes and Seychelles, to Malesia and New Caledonia. A further species, Tetraria borneensis, has been suggested to belong to Costularia. Relationships and divergence times were inferred using an existing four marker phylogeny of Cyperaceae tribe Schoeneae expanded with newly generated sequence data mainly for Costularia s.l. species. Phylogenetic reconstruction was executed using Bayesian inference and maximum likelihood approaches. Divergence times were estimated using a relaxed molecular clock model, calibrated with fossil data. Based on our results, Tetraria borneensis is not related to the species of Costularia. Costularia s.l. is composed of four distinct evolutionary lineages. Two lineages, one including the type species, are part of the Oreobolus clade, i.e. a much reduced genus Costularia restricted to southeastern Africa, Madagascar, the Mascarenes and Seychelles, and a small endemic genus from New Caledonia for which a new genus Chamaedendron is erected based on Costularia subgenus Chamaedendron. The other two lineages are part of the Tricostularia clade, i.e. a separate single-species lineage from the Seychelles for which a new genus (Xyroschoenus) is described, and Costularia subgenus Lophoschoenus. For the latter, more research is needed to test whether they are congeneric with the species placed in the reticulate-sheathed Tetraria clade.