High throughput shotgun sequencing of eRNA reveals taxonomic and derived functional shifts across a benthic productivity gradient

Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates <1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. While DNA sequencing captures both alive and dead organisms, environmental RNA (eRNA) better targets living organisms or organisms of recent origin in the environment. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in (i) biodiversity of multiple microeukaryotic communities; and (ii) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2%-6.9%), when compared to low OC sediments (1.2%-2.8%). Dissimilarity in community composition increased for all three groups between Low OC and High OC, as well as the classified feeding type of nematode genera (with more nonselective deposit feeders in high OC sediment). High relative abundant genera included nematodeSabatieriaand foraminiferaElphidiumin high OC, andCryptocaryon-like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of benthic microeukaryotes could be a powerful tool in recurring monitoring programmes.Peer reviewe

Limited congruence exhibited across microbial, meiofaunal and macrofaunal benthic assemblages in a heterogeneous coastal environment

One of the most common approaches for investigating the ecology of spatially complex environments is to examine a single biotic assemblage present, such as macroinvertebrates. Underlying this approach are assumptions that sampled and unsampled taxa respond similarly to environmental gradients and exhibit congruence across different sites. These assumptions were tested for five benthic groups of various sizes (archaea, bacteria, microbial eukaryotes/protists, meiofauna and macrofauna) in Plymouth Sound, a harbour with many different pollution sources. Sediments varied in granulometry, hydrocarbon and trace metal concentrations. Following variable reduction, canonical correspondence analysis did not identify any associations between sediment characteristics and assemblage composition of archaea or macrofauna. In contrast, variation in bacteria was associated with granulometry, trace metal variations and bioturbation (e.g. community bioturbation potential). Protists varied with granulometry, hydrocarbon and trace metal predictors. Meiofaunal variation was associated with hydrocarbon and bioturbation predictors. Taxon turnover between sites varied with only three out of 10 group pairs showing congruence (meiofauna-protists, meiofauna-macrofauna and protists-macrofauna). While our results support using eukaryotic taxa as proxies for others, the lack of congruence suggests caution should be applied to inferring wider indicator or functional interpretations from studies of a single biotic assemblage

Large benthic foraminifera under the pipette:Method development and ecological assessment in the Spermonde Archipelago

Coral reefs are described as the rainforest of the seas, because they form a marine hotspot of biodiversity. Some coral reefs live and thrive in murky waters; they are important to study because they might provide refugia for corals and other reef organisms in face of climatic instability, especially the rise of sea water temperature. To assess this concept and to monitor the dynamics of coral reefs over time and space, multiple methods were developed, including the use of bioindicator species. Large benthic foraminifera (LBF), millimetre-sized unicellular marine protists, has proven to be very useful in assessing environmental conditions in coral reefs. Compiling 25 years of research from turbid coral reefs located in the Spermonde Archipelago, South Sulawesi, Indonesia, I answer three main questions: 1) Can LBF communities be used as a warning signal for reef benthic community shift?2) Can molecular methods be used to assess LBF community compositions reliably?3) Which environmental factors trigger the spatial distribution of LBF species?This work contributes to the greater understanding of the ecology of LBF by the means of morphological and molecular data analysis. Building upon thirty years of pioneer molecular research in foraminifera, I demonstrated the strength of LBF in monitoring reefs using molecular methods in a quantitative manner. Finally, I underscored how the combination of multiple LBF taxa provide complementary information on the reef environmental conditions in terms of water quality and substrate characteristics. This information is essential to picture ancient reefs and to anticipate future changes of present coral reefs

Large benthic foraminifera under the pipette:Method development and ecological assessment in the Spermonde Archipelago

Coral reefs are described as the rainforest of the seas, because they form a marine hotspot of biodiversity. Some coral reefs live and thrive in murky waters; they are important to study because they might provide refugia for corals and other reef organisms in face of climatic instability, especially the rise of sea water temperature. To assess this concept and to monitor the dynamics of coral reefs over time and space, multiple methods were developed, including the use of bioindicator species. Large benthic foraminifera (LBF), millimetre-sized unicellular marine protists, has proven to be very useful in assessing environmental conditions in coral reefs. Compiling 25 years of research from turbid coral reefs located in the Spermonde Archipelago, South Sulawesi, Indonesia, I answer three main questions: 1) Can LBF communities be used as a warning signal for reef benthic community shift?2) Can molecular methods be used to assess LBF community compositions reliably?3) Which environmental factors trigger the spatial distribution of LBF species?This work contributes to the greater understanding of the ecology of LBF by the means of morphological and molecular data analysis. Building upon thirty years of pioneer molecular research in foraminifera, I demonstrated the strength of LBF in monitoring reefs using molecular methods in a quantitative manner. Finally, I underscored how the combination of multiple LBF taxa provide complementary information on the reef environmental conditions in terms of water quality and substrate characteristics. This information is essential to picture ancient reefs and to anticipate future changes of present coral reefs

Translating environmental DNA for monitoring nature: species, ecosystems, policy and practice

The unprecedented biodiversity loss driven by human activity is pushing ecosystems toward collapse, threatening global economies, human health, and essential natural systems that sustain life on Earth. Restoring nature requires decisions grounded in science and evidence, gained through monitoring practices. Monitoring ecosystems is essential for guiding environmental management, but traditional biomonitoring often falls short due to time-consuming, labour-intensive methods reliant on bio-indicators and limited by an underpinning of ecosystem function and services. Environmental DNA (eDNA) is revolutionising our ability to efficiently assess biodiversity, from a single species to whole communities across the Tree-of-Life. While eDNA research is advancing rapidly, its uptake in applied sectors is lagging, where it could significantly enhance the scale, speed and sensitivity of biomonitoring. This thesis explores the gap between research and application through stakeholder engagement, develops practical guidance, and conducts primary research on policy-relevant monitoring targets, including invasive species and pathogens. Stakeholder interviews across policy, industry and academia highlight the need for improved communication tools, robust validation and standardisation of methods. These views were considered during the development of guidance for DNA monitoring with citizen science. These insights also shaped the development of a quantitative PCR (qPCR) assay for detecting the invasive Chinese mitten crab (Eriocheir sinensis), which demonstrates rigorous experimental validation using a standardised scale. Enhanced ecological assessments should integrate the suite of eDNA tools, from species to whole communities, as the efficiency of eDNA pipelines enables sampling across the continuum of eDNA to host-tissue, as explored by literature review. The molecular detection of the parasite Bonamia ostreae in native and invasive oysters will aid the Solent Oyster Restoration Project by furthering the understanding of disease spread. In summary, this thesis provides a framework for integrating enhanced eDNA-based ecological assessments into policy and practice, providing tools to inform environmental management and nature restoration

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.info:eu-repo/semantics/publishedVersio

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/ or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties inmorphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper,we review the structure of conventional biotic indices, andwe present the results of pilotmetabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices.We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring program

Can aquaculture impact the surrounding biodiversity? A metabarcoding assessment

World’s population growth and rise in food consumption per capita have led to increased food demand and overexploitation of natural resources in recent decades. Such increase has threatened the global feeding schemes to maintain a balance between food supply and demand. Although “The Blue Revolution” promised to fill such gap and simultaneously alleviate the overexploitation of the oceans, deterioration of biota in the surrounding marine environment from aquaculture pollution has been reported. To investigate the effects of this pollution in the biodiversity of benthic communities, I applied a metabarcoding surveillance method before and after the establishment of a salmon aquaculture facility at Dyrøya Island, Norway. Twelve monitoring stations were established and divided into three transects, each containing four stations at increasing distance from the cages. To distinguish the patterns of impact, I estimated alpha and beta diversity for each station using two metabarcoding markers (COI and 18S). Analysis showed a significant increase of alpha biodiversity after the establishment of the aquaculture where such increase occurred only in the North transect (aligned with the main current) at all distances from the cages. Alpha diversity analysis suggested that the spread of impact was heterogeneous throughout the transects and homogeneous throughout the sampled distances. Significant differences in community composition and beta diversity (only for COI marker) after the establishment of the aquaculture were observed. The spread of such change occurred homogeneously among all the monitoring stations, transects and distances from the cages. These findings support the hypothesis that the establishment of the aquaculture activities, alone, did not lead to these changes in beta diversity, which could rather be a result of seasonal variability. This study stresses the need for high sequencing depth, broad study area, and a combination of traditional surveys with metabarcoding approaches when conducting molecular biodiversity assessments

Molecular Biodiversity of Foraminifera

Foraminifera are a diverse clade of mostly shell-building single-celled organisms. Estimation of foraminiferal diversity is critical for understanding past and present climatic conditions, as they are highly sensitive to environmental perturbations. Biodiversity estimates of foraminifera began with the counting of test (i.e., shell) microfossils composed of calcium carbonate, as they are well preserved in sediment samples. However, this view has changed with molecular biodiversity estimates, which suggest that early-diverging single-chamber (i.e., monothalamid ) species that lack preservation ability are more diverse than anticipated. Although biodiversity estimates of foraminifera at the molecular level have changed our perceptions, they possess various challenges, especially with metabarcoding approaches. The metabarcoding approach is challenging in foraminifera because small subunit ribosomal (SSU) rRNA gene does not PCR amplify universal eukaryotic primers due to the presence of large insertions. Therefore, studies of foraminiferal diversity require targeted primers. Similarly, the pair-wise sequence similarity approach to taxonomic resolution can be problematic for Foraminifera, as fewer matching reference database exists for “monothalamids”- this requires the use of a more robust phylogeny-informed taxonomy, which provides a taxonomic identification for each sequence. Also, the appropriateness of recently developed metabarcoding tools still needs validation and comparison with clustering approaches for foraminiferal biodiversity estimation. This chapter introduces the current state of knowledge of foraminiferal biodiversity while also describing the knowledge gaps addressed in this thesis