Diversity and seasonal occurrence of haptophyta in northern South China Sea through size-fractionated metabarcoding.

Haptophyta plays a key role in marine pico-nanoeukaryote communities but information on their diversity and ecology is extremely limited. A total of 103 water samples were collected in northern South China Sea to assess the diversity of haptophyta through metabarcoding targeting 18S V4 rDNA. Furthermore, we investigated the potential genetic differentiation among seasonal occurring Phaeocystis globosa using the high resolution molecular marker pgcp1. 18S V4 rDNA metabarcoding dataset revealed 41 species of haptophytes, with 16 of them as the first record in this region. Notably, six harmful species were detected, including Chrysochromulina leadbeateri, Phaeocystis globosa, and Prymnesium parvum. The pgcp1 marker revealed two clades of Phaeocystis globosa and both of them were present around the year. Clade I was found to predominate in warm season, while Clade III tended to bloom in cold waters. Our results highlight the risk potential of harmful haptophytes in the northern South China Sea.

Deep-sea meiofaunal communities in the south-eastern Levantine basin and their shaping factors - Morphological-taxonomy-free metabarcoding approach.

The <3% dissimilar Amplicon Sequence Variant (ASV) clusters of the 18S-V4 barcode were used as species-proxies for the evaluation of ASV composition and ASV diversity indices characterizing the hitherto poorly investigated meiofaunal communities of the south-eastern part of the Levantine basin. Accompanied by abundance measurements, the relationships of these characteristics with sedimentary and bottom terrain parameters were interpreted. The construction of community composition profiles, namely ASVs' list and their estimated abundances, was done using our previously established procedure (Harbuzov et al., 2022, Marine Genomics 65, 100980), combining metabarcoding with sample reads normalization by the abundance of hard-bodied meiofaunal taxa. The study province included the 54-1418 m depth range, across vertical sub-bottom horizons ranging 0-17 cm. Oxygen, hydrogen sulfide and methane concentrations in the pore water, as well as sediment grain size spectra and sedimentary protein and carbohydrate levels, were measured, followed by an evaluation of their involvement in the shaping of the meiofaunal communities' characteristics. Community composition was generally site-and-horizon dependent and its abundance decreased with increasing bottom depth and across sub-bottom horizons, typical to benthic habitats which are nourished by organic carbon from the euphotic zone. The relatively sharply inclined continental slope bottom located in the northern part of the Israeli coast was an exception. Its meiofaunal community characteristics were speculated to be affected by intensive sediment mixing and lateral transport of food from the shelf, in addition to the effect of the euphotic zone-originated food sources.

The impact of flooding from the Minjiang River on the succession of harmful algal blooms (HABs) caused by diatoms in China's offshore waters.

This study examines diatom assemblages in the Matsu Archipelago, an area influenced by Minjiang River runoff. It focuses on harmful algal blooms (HABs) that occurred between August 2021 and July 2022. Utilizing 18S rRNA metabarcoding and microscopic analysis, we observed a significant diatom bloom during early summer runoff, peaking at 5 × 105 cells L-1. The research reveals dynamic community changes during the runoff season, with dominant genera including Pseudo-nitzschia, Chaetoceros, and Skeletonema. Skeletonema cell density correlated with NO3 levels, Chaetoceros had a slight PO4 affinity, and Pseudo-nitzschia showed a negative correlation with Skeletonema. Pseudo-nitzschia, which prefers high light and pH conditions, had notably high concentrations in the flood season and in the autumn. In both, it was dominated by potential toxin-producing species - P. multistriata and P. pungens during the flooding, and P. cuspidate in the autumn. These findings highlight the intricate relationship between diatom dynamics and environmental factors, providing essential insights for managing HABs, especially Pseudo-nitzschia species, amidst environmental changes.

Metataxomics reveals Blastocystis subtypes mixed infections in Colombian children.

Blastocystis sp., is an intestinal protist with a broad host range and a high prevalence in human populations worldwide, even in developed Western countries. The publication of conflicting evidence has divided the scientific community about the pathogenic role of this parasite. Even though, genetic studies on Blastocystis sp. revealed associations between genotypes and different pathogenic profiles. Conventionally, the detection of this parasite is based on microscopic or PCR methods, which offer meager or null performance in detecting mixed infections. In this work, we applied a metataxonomic NGS approach targeting the V4 region of the eukaryotic SSU-rRNA gene and classical phylogenetic methods. This approach allowed us to detect Blastocystis sp. in stool samples from infected children living in an urban setting in the city of Medellin attending the same daycare center. Phylogenetic analysis identified the subtypes present in the children as ST1, ST2, and ST3. Besides, mixed infections of subtypes ST1 + ST3 were spotted in 16% of the analyzed stool samples.

Secondary structure construction and molecular identification of rRNA 18S V4 region E23-5-E23-6 of parasitic lice of Hominidae.

The parasitic lice of Hominidae are a class of blood-sucking insects, having a large fragment expansion region in ribosome 18S V4 region. In this study, the value of the E23-5-E23-6 stem-loop structure in the insertion region for molecular identification of lice were explored through motif analysis and secondary structure construction. Five pubic lice samples from China were morphologically identified, and primers for the rRNA 18S V4 region were designed for molecular identification. The V4 sequence of the parasitic lice of Hominidae was retrieved from GenBank for sequence analysis. The five samples were identified as pubic lice based on V4 region, which were of the same specie but geographically different from Australian strains in Genbank, with the identity of 99.06-99.46%. Compared with the human lice, both the chimpanzee lice and pubic lice had large indel fragments in the V4 region. Comparison results showed that Muscle and MAFFT had better alignment and phylogeny results than Clustal. The large expansion region, comprising E23-5 and E23-6, was located between E23-4 and E23-7. The V4 secondary structure showed that the stem-loop structures of the lice parasitizing on pubic area, human, and chimpanzee were different in the E23-5 and E23-6, which could effectively distinguish the three parasitic lice and divide the human lice into five genotypes. This is suitable not only for the identification of three lice species in higher taxonomic ranks but also for genotype identification of human lice in lower taxonomic ranks. The difference between the stem-loop structure is more intuitive than that between the primary sequences.

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization.

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.

New barcoded primers for efficient retrieval of cercozoan sequences in high-throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts.

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost-effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new-generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%-100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.

Microbial Eukaryotes in an Arctic Under-Ice Spring Bloom North of Svalbard.

Microbial eukaryotes can play prominent roles in the Arctic marine ecosystem, but their diversity and variability is not well known in the ice-covered ecosystems. We determined the community composition of microbial eukaryotes in an Arctic under-ice spring bloom north of Svalbard using metabarcoding of DNA and RNA from the hypervariable V4 region of 18S nrDNA. At the two stations studied, the photosynthetic biomass was dominated by protists >3 µm and was concentrated in the upper 70-80 m, above the thermocline and halocline. Hierarchical cluster analyses as well as ordination analyses showed a distinct clustering of the microbial eukaryote communities according to a combination of water mass and local environmental characteristics. While samples collected in the surface mixed layer differed distinctly between the two sites, the deeper communities collected in Atlantic Water were fairly similar despite being geographically distant. The differentiation of the microbial eukaryote communities of the upper mixed water was probably driven by local development and advection, while the lack of such differentiation in the communities of Atlantic Water reflects the homogenizing effect of water currents on microbial communities.