Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free.

BACKGROUND: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. RESULTS: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing. CONCLUSION: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Integrative approach to monitoring metazoan diversity and distribution in two Mediterranean coastal sites through morphology and organismal eDNA.

Marine and coastal ecosystems respond to climate change in various ways, such as the type of ecosystem, the species composition, interactions, and distribution, and the effect of local stressors. Metazoan organisms, particularly zooplankton, are important indicators for monitoring the effects climate-driven warming in marine coastal ecosystems over the long term. In this study, the diversity and distribution of zooplankton communities in the Mediterranean Sea (Canyon Dohrn and LTER-MareChiara, Gulf of Naples), a known biodiversity and climate changes hotspot, have been assessed using the integration of morphological-based identification and organismal eDNA. Our findings showed that the multi-locus strategy including the mitochondrial cytochrome c oxidase I (COI) gene and the hypervariable region V9 of the 18S rDNA (18S V9) as targets, improved the taxonomic overview, with the COI gene being more effective than the 18S V9 region for metazoans at the species level. However, appendicularians were detected only with the 18S V9 region. Overall, organismal eDNA is a powerful approach for revealing hidden biodiversity, especially for gelatinous and meroplankton components, and provided new insights into biodiversity patterns. The ecological importance of calanoid copepods in coastal ecosystems has been confirmed. In contrast, the discovery of 13 new metazoan records in the Mediterranean Sea, including two non-indigenous copepod species, suggested that local stressors affect zooplankton community structure and resilience, highlighting the importance of biomonitoring and protecting marine coastal ecosystems.

Multi-marker DNA metabarcoding for precise species identification in ichthyoplankton samples.

Ichthyoplankton monitoring is crucial for stock assessments, offering insights into spawning grounds, stock size, seasons, recruitment, and changes in regional ichthyofauna. This study evaluates the efficiency of multi-marker DNA metabarcoding using mitochondrial cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA gene markers, in comparison to morphology-based methods for fish species identification in ichthyoplankton samples. Two transects with four coastal distance categories were sampled along the southern coast of Portugal, being each sample divided for molecular and morphological analyses. A total of 76 fish species were identified by both approaches, with DNA metabarcoding overperforming morphology-75 versus 11 species-level identifications. Linking species-level DNA identifications with higher taxonomic morphological identifications resolved several uncertainties associated with traditional methods. Multi-marker DNA metabarcoding improved fish species detection by 20-36% compared to using a single marker/amplicon, and identified 38 species in common, reinforcing the validity of our results. PERMANOVA analysis revealed significant differences in species communities based on the primer set employed, transect location, and distance from the coast. Our findings underscore the potential of DNA metabarcoding to assess ichthyoplankton diversity and suggest that its integration into routine surveys could enhance the accuracy and comprehensiveness of fish stock assessments.

Ecological genomics in the Northern krill uncovers loci for local adaptation across ocean basins.

Krill are vital as food for many marine animals but also impacted by global warming. To learn how they and other zooplankton may adapt to a warmer world we studied local adaptation in the widespread Northern krill (Meganyctiphanes norvegica). We assemble and characterize its large genome and compare genome-scale variation among 74 specimens from the colder Atlantic Ocean and warmer Mediterranean Sea. The 19 Gb genome likely evolved through proliferation of retrotransposons, now targeted for inactivation by extensive DNA methylation, and contains many duplicated genes associated with molting and vision. Analysis of 760 million SNPs indicates extensive homogenizing gene-flow among populations. Nevertheless, we detect signatures of adaptive divergence across hundreds of genes, implicated in photoreception, circadian regulation, reproduction and thermal tolerance, indicating polygenic adaptation to light and temperature. The top gene candidate for ecological adaptation was nrf-6, a lipid transporter with a Mediterranean variant that may contribute to early spring reproduction. Such variation could become increasingly important for fitness in Atlantic stocks. Our study underscores the widespread but uneven distribution of adaptive variation, necessitating characterization of genetic variation among natural zooplankton populations to understand their adaptive potential, predict risks and support ocean conservation in the face of climate change.

Rearing condition influences gene expression in postlarval American lobster (Homarus americanus).

The American lobster (Homarus americanus) is an economically important species in the western Atlantic and its climate-driven range shift northward along the east coast of the United States is well documented. The thermal tolerance of lab-reared postlarvae of this species has been extensively investigated to better understand settlement and recruitment dynamics. However, there have been few studies focused on wild-caught postlarvae, and even fewer that have analyzed lab-rearing conditions in context of diet. In this study, we investigated gene transcriptional changes in postlarvae caught in the wild, as well as postlarvae reared in the laboratory on a brine shrimp diet or a wild-sourced zooplankton diet. We found between wild-caught and brine shrimp-reared larvae 3,682 differentially expressed genes, and between wild and zooplankton-reared postlarvae 3,939 differentially expressed genes. Between the two lab-reared groups fed different diets 2,603 genes were differentially expressed. We also exposed individuals in all rearing groups to chronic temperature treatments of 8°C and 26°C and found that both temperature extremes elicit 68-95% fewer transcriptional changes in wild postlarvae compared to either lab-reared group. In wild postlarvae, we identified differential expression of transcripts within the FoxO signaling pathway, a signaling pathway with a central role in cellular physiology, as potential molecular markers for cold tolerance in the American lobster. These findings contextualize the current literature on lab-reared postlarvae as containing conservative estimates for in situ organisms and can be used to inform population distribution modeling efforts. They also provide evidence for the need to adjust lab-rearing techniques or source wild larval crustaceans to augment studies of larval biology.

Time-series metagenomics reveals changing protistan ecology of a temperate dimictic lake.

BACKGROUND: Protists, single-celled eukaryotic organisms, are critical to food web ecology, contributing to primary productivity and connecting small bacteria and archaea to higher trophic levels. Lake Mendota is a large, eutrophic natural lake that is a Long-Term Ecological Research site and among the world's best-studied freshwater systems. Metagenomic samples have been collected and shotgun sequenced from Lake Mendota for the last 20 years. Here, we analyze this comprehensive time series to infer changes to the structure and function of the protistan community and to hypothesize about their interactions with bacteria. RESULTS: Based on small subunit rRNA genes extracted from the metagenomes and metagenome-assembled genomes of microeukaryotes, we identify shifts in the eukaryotic phytoplankton community over time, which we predict to be a consequence of reduced zooplankton grazing pressures after the invasion of a invasive predator (the spiny water flea) to the lake. The metagenomic data also reveal the presence of the spiny water flea and the zebra mussel, a second invasive species to Lake Mendota, prior to their visual identification during routine monitoring. Furthermore, we use species co-occurrence and co-abundance analysis to connect the protistan community with bacterial taxa. Correlation analysis suggests that protists and bacteria may interact or respond similarly to environmental conditions. Cryptophytes declined in the second decade of the timeseries, while many alveolate groups (e.g., ciliates and dinoflagellates) and diatoms increased in abundance, changes that have implications for food web efficiency in Lake Mendota. CONCLUSIONS: We demonstrate that metagenomic sequence-based community analysis can complement existing efforts to monitor protists in Lake Mendota based on microscopy-based count surveys. We observed patterns of seasonal abundance in microeukaryotes in Lake Mendota that corroborated expectations from other systems, including high abundance of cryptophytes in winter and diatoms in fall and spring, but with much higher resolution than previous surveys. Our study identified long-term changes in the abundance of eukaryotic microbes and provided context for the known establishment of an invasive species that catalyzes a trophic cascade involving protists. Our findings are important for decoding potential long-term consequences of human interventions, including invasive species introduction. Video Abstract.

Revealing the profound influence of diapause on gene expression: Insights from the annual transcriptome of the copepod Calanus finmarchicus.

Annual rhythms are observed in living organisms with numerous ecological implications. In the zooplanktonic copepod Calanus finmarchicus, such rhythms are crucial regarding its phenology, body lipid accumulation, and global carbon storage. Climate change drives annual biological rhythms out of phase with the prevailing environmental conditions with yet unknown but potentially catastrophic consequences. However, the molecular dynamics underlying phenology are still poorly described. In a rhythmic analysis of C. finmarchicus annual gene expression, results reveal that more than 90% of the transcriptome shows significant annual rhythms, with abrupt and dramatic upheaval between the active and diapause life cycle states. This work explores the implication of the circadian clock in the annual timing, which may control epigenetic mechanisms to profoundly modulate gene expression in response to calendar time. Results also suggest an increased light sensitivity during diapause that would ensure the photoperiodic entrainment of the endogenous annual clock.

Molecular evidence for a new endemic species of Acartia (Copepoda, Calanoida) from the Southeast Pacific coast.

The loss of biodiversity in marine populations is one of the consequences of the increased events of extreme environmental conditions in the oceans, which can condition the persistence of populations to future scenarios of climate change. Therefore, it is extremely necessary to explore and monitor the genetic diversity of natural populations. In the Southeast Pacific Ocean (SEPO), specifically on the coast of Chile, the presence of the copepod Acartia tonsa has been indicated solely using morphological evidence, due to the absence of genetic information. In the present work, the genetic diversity, population structure and phylogenetic position within the genus Acartia, of populations identified morphologically as A. tonsa, was evaluated by amplification of the mitochondrial cytochrome c oxidase subunit I and nuclear marker 18 s. Our results showed that the populations identified as A. tonsa correspond to a new monophyletic group endemic to SEPO (GMYC = 1.00; PTP = 0.95). The populations showed moderate to high genetic diversity with an incipient structuring between populations and biogeographic zones. Our results suggest that despite the homogenizing effect of the Humboldt Current, isolation by distance and contrasting environmental conditions at different geographic scales have an important influence on the genetic diversity of zooplankton in the SEPO region.

Using low volume eDNA methods to sample pelagic marine animal assemblages.

Environmental DNA (eDNA) is an increasingly useful method for detecting pelagic animals in the ocean but typically requires large water volumes to sample diverse assemblages. Ship-based pelagic sampling programs that could implement eDNA methods generally have restrictive water budgets. Studies that quantify how eDNA methods perform on low water volumes in the ocean are limited, especially in deep-sea habitats with low animal biomass and poorly described species assemblages. Using 12S rRNA and COI gene primers, we quantified assemblages comprised of micronekton, coastal forage fishes, and zooplankton from low volume eDNA seawater samples (n = 436, 380-1800 mL) collected at depths of 0-2200 m in the southern California Current. We compared diversity in eDNA samples to concurrently collected pelagic trawl samples (n = 27), detecting a higher diversity of vertebrate and invertebrate groups in the eDNA samples. Differences in assemblage composition could be explained by variability in size-selectivity among methods and DNA primer suitability across taxonomic groups. The number of reads and amplicon sequences variants (ASVs) did not vary substantially among shallow (<200 m) and deep samples (>600 m), but the proportion of invertebrate ASVs that could be assigned a species-level identification decreased with sampling depth. Using hierarchical clustering, we resolved horizontal and vertical variability in marine animal assemblages from samples characterized by a relatively low diversity of ecologically important species. Low volume eDNA samples will quantify greater taxonomic diversity as reference libraries, especially for deep-dwelling invertebrate species, continue to expand.

A circumpolar study of surface zooplankton biodiversity of the Southern Ocean based on eDNA metabarcoding.

Under pressure from climate change and fishing, the Southern Ocean ecosystems have been changing. Zooplankton plays a vital role in the food web of the Southern Ocean and is crucial for maintaining ecosystem stability. Investigating the circumpolar-scale species composition and biodiversity of zooplankton is crucial for ensuring ecosystem-based conservation and management of the Southern Ocean in a changing climate. Here, we utilized eDNA metabarcoding to assess the biodiversity of zooplankton in the surface seawater surrounding the Antarctica based on samples collected during two expeditions spanning from 2021 to 2022. The main purpose of this paper is to provide more baseline information about circumpolar zooplankton biodiversity based on the emerging eDNA metabarcoding tool. This comprehensive approach led to the identification of over 300 distinct zooplankton species, forming a diverse community dominated by Jellyfish, Mollusca and Polychaete. Surprisingly, common dominant taxonomic groups such as krill and copepods in the Southern Ocean did not show high relative abundance (reads) in surface seawater. The results of redundancy analysis (RDA) and correlation analysis highlighted that water temperature and chlorophyll a had the most significant impact on the reads and diversity of zooplankton. Notably, the influence of water temperature on zooplankton seemed to be primarily indirect, potentially mediated by its effects on primary productivity. Increasing in primary production might lead to lower zooplankton biodiversity in the Southern Ocean in future. This research underscores the effectiveness of eDNA metabarcoding as a valuable tool for monitoring zooplankton diversity in open seas. Given the ongoing changes in temperature, sea ice extent and their impact on primary production, our findings lay a crucial foundation for using eDNA techniques to establish long-term biodiversity monitoring programs across extensive marine ecosystems in the future.

Unprecedented insights into extents of biological responses to physical forcing in an Arctic sub-mesoscale filament by combining high-resolution measurement approaches.

In Fram Strait, we combined underway-sampling using the remote-controlled Automated Filtration System for Marine Microbes (AUTOFIM) with CTD-sampling for eDNA analyses, and with high-resolution optical measurements in an unprecedented approach to determine variability in plankton composition in response to physical forcing in a sub-mesoscale filament. We determined plankton composition and biomass near the surface with a horizontal resolution of ~ 2 km, and addressed vertical variability at five selected sites. Inside and near the filament, plankton composition was tightly linked to the hydrological dynamics related to the presence of sea ice. The comprehensive data set indicates that sea-ice melt related stratification near the surface inside the sub-mesoscale filament resulted in increased sequence abundances of sea ice-associated diatoms and zooplankton near the surface. In analogy to the physical data set, the underway eDNA data, complemented with highly sampled phytoplankton pigment data suggest a corridor of 7 km along the filament with enhanced photosynthetic biomass and sequence abundances of sea-ice associated plankton. Thus, based on our data we extrapolated an area of 350 km2 in Fram Strait with enhanced plankton abundances, possibly leading to enhanced POC export in an area that is around a magnitude larger than the visible streak of sea-ice.

Dynamics of Gut Bacteria Across Different Zooplankton Genera in the Baltic Sea.

In aquatic ecosystems, zooplankton-associated bacteria potentially have a great impact on the structure of ecosystems and trophic networks by providing various metabolic pathways and altering the ecological niche of host species. To understand the composition and drivers of zooplankton gut microbiota, we investigated the associated microbial communities of four zooplankton genera from different seasons in the Baltic Sea using the 16S rRNA gene. Among the 143 ASVs (amplified sequence variants) observed belonging to heterotrophic bacteria, 28 ASVs were shared across all zooplankton hosts over the season, and these shared core ASVs represented more than 25% and up to 60% of relative abundance in zooplankton hosts but were present at low relative abundance in the filtered water. Zooplankton host identity had stronger effects on bacterial composition than seasonal variation, with the composition of gut bacterial communities showing host-specific clustering patterns. Although bacterial compositions and dominating core bacteria were different between zooplankton hosts, higher gut bacteria diversity and more bacteria contributing to the temporal variation were found in Temora and Pseudocalanus, compared to Acartia and Synchaeta. Diet diatom and filamentous cyanobacteria negatively correlated with gut bacteria diversity, but the difference in diet composition did not explain the dissimilarity of gut bacteria composition, suggesting a general effect of diet on the inner conditions in the zooplankton gut. Synchaeta maintained high stability of gut bacterial communities with unexpectedly low bacteria-bacteria interactions as compared to the copepods, indicating host-specific regulation traits. Our results suggest that the patterns of gut bacteria dynamics are host-specific and the variability of gut bacteria is not only related to host taxonomy but also related to host behavior and life history traits.

An in situ digital synthesis strategy for the discovery and description of ocean life.

Revolutionary advancements in underwater imaging, robotics, and genomic sequencing have reshaped marine exploration. We present and demonstrate an interdisciplinary approach that uses emerging quantitative imaging technologies, an innovative robotic encapsulation system with in situ RNA preservation and next-generation genomic sequencing to gain comprehensive biological, biophysical, and genomic data from deep-sea organisms. The synthesis of these data provides rich morphological and genetic information for species description, surpassing traditional passive observation methods and preserved specimens, particularly for gelatinous zooplankton. Our approach enhances our ability to study delicate mid-water animals, improving research in the world's oceans.

Potential PCR amplification bias in identifying complex ecological patterns: Higher species compositional homogeneity revealed in smaller-size coral reef zooplankton by metatranscriptomics.

PCR-based high-throughput sequencing has permitted comprehensive resolution analyses of zooplankton diversity dynamics. However, significant methodological issues still surround analyses of complex bulk community samples, not least as in prevailing PCR-based approaches. Marine drifting animals-zooplankton-play essential ecological roles in the pelagic ecosystem, transferring energy and elements to higher trophic levels, such as fishes, cetaceans and others. In the present study, we collected 48 size-fractionated zooplankton samples in the vicinity of a coral reef island with environmental gradients. To investigate the spatiotemporal dynamics of zooplankton diversity patterns and the effect of PCR amplification biases across these complex communities, we first took metatranscriptomics approach. Comprehensive computational analyses revealed a clear pattern of higher/lower homogeneity in smaller/larger zooplankton compositions across samples respectively. Our study thus suggests changes in the role of dispersal across the sizes. Next, we applied in silico PCR to the metatranscriptomics datasets, in order to estimate the extent of PCR amplification bias. Irrespective of stringency criteria, we observed clear separations of size fraction sample clusters in both metatranscriptomics and in silico datasets. In contrast, the pattern-smaller-fractioned communities had higher compositional homogeneity than larger ones-was observed in the metatranscriptomics data but not in the in silico datasets. To investigate this discrepancy further, we analysed the mismatches of widely used mitochondrial CO1 primers and identified priming site mismatches likely driving PCR-based biases. Our results suggest the use of metatranscriptomics or, although less ideal, redesigning the CO1 primers is necessary to circumvent these issues.

Holistic view of the seascape dynamics and environment impact on macro-scale genetic connectivity of marine plankton populations.

BACKGROUND: Plankton seascape genomics studies have revealed different trends from large-scale weak differentiation to microscale structures. Previous studies have underlined the influence of the environment and seascape on species differentiation and adaptation. However, these studies have generally focused on a few single species, sparse molecular markers, or local scales. Here, we investigated the genomic differentiation of plankton at the macro-scale in a holistic approach using Tara Oceans metagenomic data together with a reference-free computational method. RESULTS: We reconstructed the FST-based genomic differentiation of 113 marine planktonic taxa occurring in the North and South Atlantic Oceans, Southern Ocean, and Mediterranean Sea. These taxa belong to various taxonomic clades spanning Metazoa, Chromista, Chlorophyta, Bacteria, and viruses. Globally, population genetic connectivity was significantly higher within oceanic basins and lower in bacteria and unicellular eukaryotes than in zooplankton. Using mixed linear models, we tested six abiotic factors influencing connectivity, including Lagrangian travel time, as proxies of oceanic current effects. We found that oceanic currents were the main population genetic connectivity drivers, together with temperature and salinity. Finally, we classified the 113 taxa into parameter-driven groups and showed that plankton taxa belonging to the same taxonomic rank such as phylum, class or order presented genomic differentiation driven by different environmental factors. CONCLUSION: Our results validate the isolation-by-current hypothesis for a non-negligible proportion of taxa and highlight the role of other physicochemical parameters in large-scale plankton genetic connectivity. The reference-free approach used in this study offers a new systematic framework to analyse the population genomics of non-model and undocumented marine organisms from a large-scale and holistic point of view.

Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences.

Background: Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples. Methods: rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications. Results: A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas. Discussion: The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems.

Metabarcoding analysis of marine zooplankton confirms the ecological role of a sheltered bight along an exposed continental shelf.

Zooplankton plays an essential role in marine ecosystems as the link between primary producers (phytoplankton) and higher trophic levels in food webs, and as a dynamic pool of recruits for invertebrates and fish. Zooplankton communities are diverse with a patchy distribution at different spatial scales, influenced by oceanographic processes. The continental shelf of eastern South Africa is narrow and exposed to the western-boundary Agulhas Current, with some shelter against strong directional flow provided by the broader KwaZulu-Natal Bight, a coastal offset adjacent to an estuary. We compared zooplankton species richness, diversity and relative abundance of key taxa among sheltered and exposed shelf areas using metabarcoding and community analysis, to explore the ecological role of the bight in a highly dynamic ocean region. Metabarcoding recovered higher richness and diversity at a finer resolution than could previously be achieved with traditional microscopy. Of 271 operational taxonomic units (OTUs) recovered through metabarcoding, 63% could be matched with >95% sequence similarity to reference barcodes. OTUs were dominated by malacostracan crustaceans (161 spp.), ray-finned fishes (45 spp.) and copepods (28 spp.). Species richness, diversity and the relative abundance of key taxa differed between sheltered and exposed shelf areas. Lower species richness in the bight was partly attributed to structurally homogeneous benthic habitats, and an associated reduction of meroplanktonic species originating from local benthic-pelagic exchange. High relative abundance of a ray-finned fish in the bight, as observed based on fish eggs and read counts, confirmed that the bight is an important fish spawning area. Overall, zooplankton metabarcoding outputs were congruent with findings of previous ecological research using more traditional methods of observation.

The microbiome of the pelagic tunicate Dolioletta gegenbauri: A potential link between the grazing and microbial food web.

Bloom-forming gelatinous zooplankton occur circumglobally and significantly influence the structure of pelagic marine food webs and biogeochemical cycling through interactions with microbial communities. During bloom conditions especially, gelatinous zooplankton are keystone taxa that help determine the fate of primary production, nutrient remineralization, and carbon export. Using the pelagic tunicate Dolioletta gegenbauri as a model system for gelatinous zooplankton, we carried out a laboratory-based feeding experiment to investigate the potential ecosystem impacts of doliolid gut microbiomes and microbial communities associated with doliolid faecal pellets and the surrounding seawater. Metabarcoding targeting Bacteria and Archaea 16S rRNA genes/Archaea) and qPCR approaches were used to characterize microbiome assemblages. Comparison between sample types revealed distinct patterns in microbial diversity and biomass that were replicable across experiments. These observations support the hypothesis that through their presence and trophic activity, doliolids influence the structure of pelagic food webs and biogeochemical cycling in subtropical continental shelf systems where tunicate blooms are common. Bacteria associated with starved doliolids (representative of the resident gut microbiome) possessed distinct low-biomass and low-diversity microbial assemblages, suggesting that the doliolid microbiome is optimized to support a detrital trophic mode. Bacterial genera Pseudoalteromomas and Shimia were the most abundant potential core microbiome taxa, similar to patterns observed in other marine invertebrates. Exploratory bioinformatic analyses of predicted functional genes suggest that doliolids, via their interactions with bacterial communities, may affect important biogeochemical processes including nitrogen, sulphur, and organic matter cycling.

DNA metabarcoding reveals impact of local recruitment, dispersal, and hydroperiod on assembly of a zooplankton metacommunity.

Understanding the environmental impact on the assembly of local communities in relation to their spatial and temporal connectivity is still a challenge in metacommunity ecology. This study aims to unravel underlying metacommunity processes and environmental factors that result in observed zooplankton communities. Unlike most metacommunity studies, we jointly examine active and dormant zooplankton communities using a DNA metabarcoding approach to overcome limitations of morphological species identification. We applied two-fragment (COI and 18S) metabarcoding to monitor communities of 24 kettle holes over a two-year period to unravel (i) spatial and temporal connectivity of the communities, (ii) environmental factors influencing local communities, and (iii) dominant underlying metacommunity processes in this system. We found a strong separation of zooplankton communities from kettle holes of different hydroperiods (degree of permanency) throughout the season, while the community composition within single kettle holes did not differ between years. Species richness was primarily dependent on pH and permanency, while species diversity (Shannon Index) was influenced by kettle hole location. Community composition was impacted by kettle hole size and surrounding field crops. Environmental processes dominated temporal and spatial processes. Sediment communities showed a different composition compared to water samples but did not differ between ephemeral and permanent kettle holes. Our results suggest that communities are mainly structured by environmental filtering based on pH, kettle hole size, surrounding field crops, and permanency. Environmental filtering based on specific conditions in individual kettle holes seems to be the dominant process in community assembly in the studied zooplankton metacommunity.

Using DNA Metabarcoding to Characterize the Prey Spectrum of Two Co-Occurring Themisto Amphipods in the Rapidly Changing Atlantic-Arctic Gateway Fram Strait.

The two congeneric hyperiids Themisto libellula and T. abyssorum provide an important trophic link between lower and higher trophic levels in the rapidly changing Arctic marine ecosystem. These amphipods are characterized by distinct hydrographic affinities and are hence anticipated to be impacted differently by environmental changes, with major consequences for the Arctic food web. In this study, we applied DNA metabarcoding to the stomach contents of these Themisto species, to comprehensively reveal their prey spectra at an unprecedented-high-taxonomic-resolution and assess the regional variation in their diet across the Fram Strait. Both species feed on a wide variety of prey but their diet strongly differed in the investigated summer season, showing overlap for only a few prey taxa, such as calanoid copepods. The spatially structured prey field of T. libellula clearly differentiated it from T. abyssorum, of which the diet was mainly dominated by chaetognaths. Our approach also allowed the detection of previously overlooked prey in the diet of T. libellula, such as fish species and gelatinous zooplankton. We discuss the reasons for the differences in prey spectra and which consequences these may have in the light of ongoing environmental changes.