Metatranscriptome of a marine pelagic crustacean community using nanopore sequencing

Due to their rapid responses to environmental variation, planktonic organisms are used as bio-indicators of ecosystem changes. Most zooplankton monitoring studies focus mainly on variability in biodiversity, densities and biomass. Advances in practical, cost-effective molecular approaches can help overcome the issues with morphology-based biomonitoring. While molecular studies are growing in popularity, a fundamental challenge remains the transport of biological material to a laboratory for DNA/RNA extractions and sequencing. The MinION™, a portable nanopore-based DNA/RNA sequencing platform (Oxford Nanopore Technologies), offers big potential advantages in the context of biodiversity research, i.e. portability and low costs of instrument and reagents. It weighs less than 100 g, is therefore easily transportable and is powered to sequence RNA using the USB port on a standard laptop, hence making it suitable for mobile research setups and real time monitoring campaigns onsite. In a first step, we wanted to understand how the gene expression in zooplankton fluctuates over the course of a short time period. Therefore, we monitored the gene expression of the dominant zooplankter, the calanoid copepod Temora longicornis, over a short time span (one day), making use of the infrastructure of the research vessel Simon Stevin. We compared gene expression results with in situ determined biotic and abiotic patterns. Moreover, due to the recent development of the VolTRAX, a small device designed to perform library preparation automatically, for the first time we were able to prepare a biological sample for analysis in situ and hands-free, making in-field, molecular monitoring of marine life possible

Metabarcoding of marine zooplankton communities in the North Sea using nanopore sequencing

Zooplankton are crucial organisms both in terms of biodiversity and their unique position in aquatic food webs. As such, it is crucial that we improve our insights into how anthropogenic and natural factors may affect these pelagic organisms. Although easily collected in large numbers, the subsequent processing and identification of specimens has usually been a barrier to large-scale biodiversity assessments. DNA barcoding, the use of standardized short gene regions to discriminate species, has been increasingly used by non-taxonomists to identify species. Here, we measured the diversity and community composition of zooplankton in the Belgian part of the North Sea over the course of one year. We identified zooplankton using both a traditional approach, based on morphological characteristics, and by metabarcoding of a 650 bp fragment of the 18S rRNA gene using the MinION™, a portable nanopore-based DNA sequencing platform. We established a method for characterizing zooplankton communities in marine samples using nanopore sequencing. We were able to identify several taxa at the species level, across a broad taxonomic scale and we thus could obtain several diversity metrics, allowing comparisons of diversity and community composition

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

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

Investigating taxon boundaries and extinction risk in endemic Chilean cacti (Copiapoa subsection Cinerei, Cactaceae) using chloroplast DNA sequences, microsatellite data and 3D mapping

Copiapoa (Cactaceae) is a genus endemic to the Chilean Atacama Desert. The taxa of Copiapoa subsection Cinerei occur in an area of high species richness and high levels of species endemism of the Central Chilean biodiversity hotspot. Four taxa are usually recognised in this group: Copiapoa gigantea (sometimes placed in C. cinerea as C. cinerea subsp. haseltoniana) and C. cinerea including three subspecies (subsp. cinerea, subsp. krainziana and subsp. columna-alba), one of which is often recognised at species level, i.e. the narrow endemic C. krainziana. Here, we evaluate the taxon boundaries of Copiapoa subsection Cinerei using chloroplast sequences and microsatellite data. We generated sequences of three cpDNA markers (rpl32–trnL, trnH–psbA, ycf1) and as indicated in a previous study, found variation between C. gigantea and C. cinerea on a subsample of 34 individuals. Five microsatellite loci were genotyped for 68 individuals from the known range of Copiapoa subsection Cinerei. In contrast with expectations, we found relatively high levels of genetic diversity (e.g., He = 0.775 – 0.827; Ho = 0.580 – 0.750) and no population structure, even between the two species. Additionally, species distribution models were conducted based on abiotic suitability and transformed to 3D maps to account for topographical complexity. The species distribution models and their 3D projections support an allopatric distribution of the four taxa of Copiapoa subsection Cinerei, with each taxon related to a different range with complex topographical features. The obtained molecular results, combined with the presented species distribution modelling, and calculations of extent of occurrence and area of occupancy for the four taxa of Copiapoa subsection Cinerei, suggest a high extinction risk for most of the taxa. A taxonomic treatment is provided

Targeted sequencing supports morphology and embryo features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus. Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted sequencing in order to: (1) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae and Cypereae; (2) define the limits of Fuireneae s.s., and (3) test the monophyly of Fuireneae s.l. genera with emphasis on Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses strongly support six genera and four major Fuireneae s.l. clades that we recognise as tribes: Bolboschoeneae stat.nov., Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr.nov. These results are consistent with morphological, micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level, most sub‐Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella. Our targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognised tribes and genera are provided

PERBEDAAN TINGKAT PENGETAHUAN DAN SIKAP IBU HAMIL SEBELUM DAN SESUDAH PENYULUHAN MENGENAI IUD PASCAPLASENTA

Background : Post-placental IUD is defined as IUD insertion within 10 minutes after the expulsion of placenta. This method is considered safe and the most potential method to prevent missed opportunity. The use of post-placental IUD currently perceived incompatible with the expectation. One of the factors that cause this thing is the low knowledge level of post-placental IUD in society. Aim : To analyze the difference of knowledge level and attitude towards postplacental IUD on pregnant woman before and after counseling. Methods : This research was conducted using quasi experimental one group pretest-posttest design. Thirty-three samples were taken by consecutive sampling from pregnant woman who lived in the territory of Puskesmas Ngesrep and Puskemas Halmahera, Semarang. Data were analyzed using paired T-Test. Results : The mean value of knowledge level before counseling was 6,09 ± 1,826 whereas after counseling was 8,15 ± 1,661. The mean value of attitude before counseling was 35,79 ± 4,709 with 26 respondents had positive attitude and 7 respondents had negative attitude whereas after counseling the mean value was 39,97 ± 4,261 with all respondents had positive attitude. There was significant difference (p<0,05) on knowledge level (p=0,000) and attitude (p=0,000) before and after counseling. Conclusion : There was significant difference on knowledge level and attitude towards post-placental IUD on pregnant woman before and after counseling. Key words: Post-placental IUD, counseling, knowledge level, attitud

A new classification of Cyperaceae (Poales) supported by phylogenomic data

Cyperaceae (sedges) are the third largest monocot family and are of considerable economic and ecological importance. Sedges represent an ideal model family to study evolutionary biology because of their species richness, global distribution, large discrepancies in lineage diversity, broad range of ecological preferences, and adaptations including multiple origins of C4 photosynthesis and holocentric chromosomes. Goetghebeur’s seminal work on Cyperaceae published in 1998 provided the most recent complete classification at tribal and generic level, based on a morphological study of Cyperaceae inflorescence, spikelet, flower and embryo characters plus anatomical and other information. Since then, several family‐level molecular phylogenetic studies using Sanger sequence data have been published. Here, more than 20 years after the last comprehensive classification of the family, we present the first family‐wide phylogenomic study of Cyperaceae based on targeted sequencing using the Angiosperms353 probe kit sampling 311 accessions. Additionally, 62 accessions available from GenBank were mined for overlapping reads and included in the phylogenomic analyses. Informed by this backbone phylogeny, a new classification for the family at the tribal, subtribal and generic levels is proposed. The majority of previously recognized suprageneric groups are supported, and for the first time we establish support for tribe Cryptangieae as a clade including the genus Koyamaea. We provide a taxonomic treatment including identification keys and diagnoses for the 2 subfamilies, 24 tribes and 10 subtribes and basic information on the 95 genera. The classification includes five new subtribes in tribe Schoeneae: Anthelepidinae, Caustiinae, Gymnoschoeninae, Lepidospermatinae and Oreobolinae. This article is protected by copyright. All rights reserved

Temporal effects of chemical and physical stressors on marine zooplankton : a molecular approach

Oceans and seas are often perceived as the last wilderness on the planet. However, anthropogenic actions are already impacting these ecosystems, ranging from the coasts and the sea surface to the open ocean and the deep sea floor. Marine ecosystems are currently affected by multiple human activities, such as eutrophication, overfishing, the introduction of non-indigenous species, the contamination by hazardous chemicals and (micro)plastics, etc., in addition to climate change, leading to impaired environmental conditions. Evidence is growing that these changing environmental conditions have negative effects on the biodiversity and functioning of marine food webs. Due to their rapid responses to environmental variation, planktonic organisms are used as bio-indicators of ecosystem changes. With the need for better understanding the impact of a changing environment on zooplankton communities, zooplankton monitoring programs have been carried out in the marine environment globally since the early 20th century. Most zooplankton monitoring studies focus mainly on variability in biodiversity and biomass. However, this approach is hindered by challenges in the identification, which is time-consuming, complicated and requires biological expertise. A combination of new technologies and techniques, together with classical in situ and laboratory studies, could improve our understanding of such biodiversity patterns by assessing the health and physiology of marine plankton. In this thesis, we aimed to apply molecular methods to investigate spatiotemporal patterns in zooplankton dynamics, as well as to investigate the influence of environmental variation and stressors on these dynamics. In chapter 2 of the thesis, we examined the spatial and temporal distribution of the zooplankton assemblage of the Belgian Part of the North Sea (BPNS) during a one-year period using both a metabarcoding approach as well as the traditional (microscopy) approach. A 650 bp fragment of the V4 and V5 region of the 18S rDNA barcode was characterized using the MinION™, a nanopore-based DNA/RNA sequencing platform (Oxford Nanopore Technologies). Metabarcoding allowed for comparisons of diversity and community composition, but not all groups (cumaceans, harpacticoid copepods) were successfully recovered. Additionally, some disparities existed between relative abundances of the most abundant taxa based on traditional counts and those based on sequence reads. Overall, we conclude that for zooplankton samples, metabarcoding is capable of detecting taxa with a higher resolution compared to microscopy, regardless of the developmental stage of the organism. A combination of molecular and morphological methods results in the highest detection and identification levels of zooplankton. The majority of the sequenced reads could be assigned to five taxa, i.e. the calanoid copepods Temora longicornis, Acartia clausi, Centropages sp., Calanus helgolandicus and Paracalanus parvus. A more comprehensive molecular data set would be able to identify and assess the impact of the main drivers of changes in the marine ecosystem, rather than only determining species richness. Studying the functional activities of a community - in situ and without a priori knowledge of genes - has been facilitated by metatranscriptomics, i.e. the study of community gene transcription. Therefore, in chapter 3, we describe and evaluate the construction of a metatranscriptome dataset from the pelagic crustacean zooplankton community, sampled in one marine station in both winter and summer. We generated transcripts using the MinION, a portable, real-time DNA and RNA sequencing device. We found that metatranscriptomics is also capable of species detection, mainly identifying calanoid copepods, particularly Temora longicornis and Acartia clausi. GO term annotation revealed that genes involved in glycolytic and translation-related processes were most expressed in the community. Based on the results of the previous chapters, T. longicornis appears to be the most dominant zooplankton species in the BPNS. Despite its economic and ecological importance, 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, as described in chapter 4. After both an acute (1 day) and a more sustained (5 days) thermal exposure to 17 °C, we investigated gene expression differences compared to animals exposed to 14 °C. 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 - 9 - 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. Given the uncertainties regarding the molecular mechanisms involved in physiological and behavioural adaptation, the goal of chapter 5 was to explore shifts in gene expression in a population of adult T. longicornis in the BPNS, collected at different time points within 24 hours and within one week. Using Direct RNA Sequencing (dRNA seq), we generated approximately 2.5 million high quality reads with the MinION™. Differentially expressed gene (DEG) analysis of field collected adults identified up to 254 significant differences in gene expression, when comparing samples taken in the evening and later at night. Our results indicate that copepods use cuticular and metabolic transcripts as a molecular mechanism to compensate for alternating conditions. We also found that biological processes such as regulation of the plasma membrane, translation, transport and signal transduction were significantly different represented in our dataset, as confirmed by enrichment and network analyses. We did not find any significant differences in gene expression in transcripts involved in the core circadian machinery of T. longicornis, probably to limitations in the sequencing depth. In chapter 6, we explored variation in population gene transcription across time and space using T. longicornis samples, collected at four different locations in the 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 were 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. No clear spatial variation in expression patterns was found based on this dataset. In chapter 7, we investigated the relative contribution of environmental variables to the densities, biomass and gene expression of Temora longicornis, based on a 4 year sampling campaign. We found spatial variation in the population density, as well as in body size, comparing copepods collected in the nearshore station as compared to the more offshore sampling stations. We applied generalized additive models to quantify the relative contribution of temperature, nutrients, salinity, turbidity, photosynthetic pigment concentrations and chemical pollution (i.e. polychlorinated biphenyls and polycyclic aromatic hydrocarbons) to the density and biomass dynamics of this species. Comparing both GAM and molecular methods, the same environmental parameters emerge influencing T. longicornis densities, biomass and gene expression (i.e. Temperature, salinity, turbidity, summed PAH concentrations). Temperature was the most important environmental variable predicting the abundances and biomass of T. longicornis. The relative contributions of turbidity, salinity and summed PAH concentrations were rather modest. Studying the gene expression of field collected adults, we identified significant differences in expression of genes involved in metabolic processes and response to stressors. We found significant correlations between temperature and genes involved in vitellogenin production, proteolytic activities, heat shock proteins. The measured anthropogenic chemical concentrations did not induce significant differences in the gene expression of typical stress related genes, such as glutathione transferases or cytochrome P450. This study underlines the potential of field gene expression studies for biomonitoring purposes and the significance of considering seasonal variation in future studies. In chapter 8, we combined and compared copepod abundance data of four dominant calanoid and the dominant harpacticoid copepod species collected in the BPNS during 2018–2021 with previously collected (2009–2010, 2015-2016) datasets for the same study area. The time series revealed a significant decrease in calanoid copepod abundance (Temora longicornis, Acartia clausi, Centropages sp., Calanus helgolandicus), while this was not the case for the studied harpacticoid copepod species, Euterpina acutifrons. We applied generalized additive models to quantify the relative contribution of temperature, nutrients, salinity, turbidity and anthropogenic chemicals (i.e. polychlorinated biphenyls and polycyclic aromatic hydrocarbons) to the dynamics of these species. Temperature was the only predictor consistently showing a high importance in all models predicting the abundances of the selected species. The various heat waves during the summer periods of these years are considered potential causes for these copepod decreases, since they corresponded to the physiological thermal limit of some of the studied copepod species. The results from this study illustrate the changes affecting this essential trophic level and highlights the value and relevance of biomonitoring and the collection of long-term data series in the context of climate change and water quality

Temporal effects of chemical and physical stressors on marine zooplankton : a molecular approach

Oceans and seas are often perceived as the last wilderness on the planet. However, anthropogenic actions are already impacting these ecosystems, ranging from the coasts and the sea surface to the open ocean and the deep sea floor. Marine ecosystems are currently affected by multiple human activities, such as eutrophication, overfishing, the introduction of non-indigenous species, the contamination by hazardous chemicals and (micro)plastics, etc., in addition to climate change, leading to impaired environmental conditions. Evidence is growing that these changing environmental conditions have negative effects on the biodiversity and functioning of marine food webs. Due to their rapid responses to environmental variation, planktonic organisms are used as bio-indicators of ecosystem changes. With the need for better understanding the impact of a changing environment on zooplankton communities, zooplankton monitoring programs have been carried out in the marine environment globally since the early 20th century. Most zooplankton monitoring studies focus mainly on variability in biodiversity and biomass. However, this approach is hindered by challenges in the identification, which is time-consuming, complicated and requires biological expertise. A combination of new technologies and techniques, together with classical in situ and laboratory studies, could improve our understanding of such biodiversity patterns by assessing the health and physiology of marine plankton. In this thesis, we aimed to apply molecular methods to investigate spatiotemporal patterns in zooplankton dynamics, as well as to investigate the influence of environmental variation and stressors on these dynamics