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

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

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

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