Diatom DNA Research Articles

Using DNA metabarcoding to characterize national scale diatom-environment relationships and to develop indicators in streams and rivers of the United States

Recent advancements in DNA techniques, metabarcoding, and bioinformatics could help expand the use of benthic diatoms in monitoring and assessment programs by providing relatively quick and increasingly cost-effective ways to quantify diatom diversity in environmental samples. However, such applications of DNA-based approaches are relatively new, and in the United States, unknowns regarding their applications at large scales exist because only a few small-scale studies have been done. Here, we present results from the first nationwide survey to use DNA metabarcoding (rbcL) of benthic diatoms, which were collected from 1788 streams and rivers across nine ecoregions spanning the conterminous USA. At the national scale, we found that diatom assemblage structure (1) was strongly associated with total phosphorus and total nitrogen concentrations, conductivity, and pH and (2) had clear patterns that corresponded with differences in these variables among the nine ecoregions. These four variables were strong predictors of diatom assemblage structure in ecoregion-specific analyses, but our results also showed that diatom-environment relationships, the importance of environmental variables, and the ranges of these variables within which assemblage changes occurred differed among ecoregions. To further examine how assemblage data could be used for biomonitoring purposes, we used indicator species analysis to identify ecoregion-specific taxa that decreased or increased along each environmental gradient, and we used their relative abundances of gene reads in samples as metrics. These metrics were strongly correlated with their corresponding variable of interest (e.g., low phosphorus diatoms with total phosphorus concentrations), and generalized additive models showed how their relationships compared among ecoregions. These large-scale national patterns and nine sets of ecoregional results demonstrated that diatom DNA metabarcoding is a robust approach that could be useful to monitoring and assessment programs spanning the variety of conditions that exist throughout the conterminous United States.

Molecular metrics to monitor ecological status of large rivers: Implementation of diatom DNA metabarcoding in the Joint Danube Survey 4

The Joint Danube Survey (JDS) is a regular transnational survey to monitor the quality of the Danube and its main tributaries, in accordance with the EU Water Framework Directive. During the JDS4 in 2019, conventional methods to monitor selected biological quality elements were complemented with DNA metabarcoding.All together 72 phytobenthos samples were collected along the Danube and its major tributaries within the JDS4, using light microscopy and DNA metabarcoding amplifying a fragment of the rbcL marker gene. The (i) applicability of DNA metabarcoding to identify diatom communities compared to microscopy; (ii) diversity metrics between DNA metabarcoding and microscopy analysis and (iii) the usability of DNA metabarcoding for routine monitoring and assessment of the Danube under future JDS surveys were investigated.Diatom communities resulting from light microscopy and DNA metabarcoding assessments share 26.5% of all taxa which corresponds to 64.3% when considering relative abundances. Discrepancies originate from biases both from metabarcoding, e.g. missing taxa from the reference library, and from microscopy, e.g. overlooking of hardly visible taxa. Microscopy detected more taxa in total but metabarcoding revealed a higher alpha diversity, detecting also very rare taxa in a given sample. Molecular and microscopy based Specific Pollution Sensitivity Index (IPS) values correlated significantly but differences were detected at several sites due to the differences in community composition and the overestimation of large taxa by metabarcoding. Although both methods showed a decreasing trend of IPS along the Danube, the metabarcoding based IPS covered a higher range of quality classes indicating lower values for downstream sites and the tributaries.We suggest that metabarcoding provides a standardisable and efficient tool in biomonitoring, being more distinctive among quality classes than microscopy. Due to the high sequencing depth, it is able to detect a higher diversity on genetic level in a time- and cost-efficient manner that should be implemented in future quality assessment tools. We recommend its use in future biomonitoring surveys, for now, as a complementary method to conventional ones.

Extraction of Diatom DNA from Water Samples and Tissues.

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Metabarcoding of microeukaryotes in surface sediments from the Pacific Arctic and adjacent sea areas: The role of diatoms in the biological pump

To better understand the importance of diatoms in carbon burial and biological pumps in the Pacific Arctic, we analyzed the microeukaryotic assemblages in twenty-six surface sediments from the northern Bering and Chukchi Seas and adjacent sea areas by metabarcoding the V4 region of the 18S rRNA gene. Biogenic elements were measured, and the contributions of diatoms to sediment carbon are discussed. A total of 618 operational taxonomic units (OTUs) of microeukaryotes were identified. Microalgae accounted for a majority of the DNA reads. Diatoms predominated in the microeukaryotic sequences, and Chaetoceros socialis was the most dominant species, especially in the Bering and Chukchi Seas. The microeukaryotic community was different in the northern deep sea, as represented by the decrease in algal and diatom reads. Organic carbon mainly originated from marine sources in the Chukchi Sea, while the Bering Sea and northern deep seas had relatively more terrestrial inputs based on the TOC/TN data. High BSi concentrations (average > 5% with a maximum of 12.87%) and BSi/TOC ratios (average > 2%) in the Bering and Chukchi Seas indicated the high diatom productivity and efficiency of the silicate pump. Diatom DNA reads were positively correlated with all biogenic elements, indicating that diatoms play an important role in organic matter burial, especially for the dominant diatom species C. socialis due to its fast growth rate and bloom- and spore-forming abilities.

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities

Little is still known about the low dose effects of radiation on the microbial communities in the environment. Mineral springs are ecosystems than can be affected by natural radioactivity. These extreme environments are, therefore, observatories for studying the influence of chronic radioactivity on the natural biota. In these ecosystems we find diatoms, unicellular microalgae, playing an essential role in the food chain. The present study aimed to investigate, using DNA metabarcoding, the effect of natural radioactivity in two environmental compartments (i.e. spring sediments and water) on the genetic richness, diversity and structure of diatom communities in 16 mineral springs in the Massif Central, France. Diatom biofilms were collected during October 2019, and a 312 bp region of the chloroplast gene rbcL (coding for the Ribulose Bisphosphate Carboxylase) used as a barcode for taxonomic assignation. A total of 565 amplicon sequence variants (ASV) were found. The dominant ASV were associated with Navicula sanctamargaritae, Gedaniella sp., Planothidium frequentissimum, Navicula veneta, Diploneis vacillans, Amphora copulata, Pinnularia brebissonii, Halamphora coffeaeformis, Gomphonema saprophilum, and Nitzschia vitrea, but some of the ASVs could not be assigned at the species level. Pearson correlation failed to show a correlation between ASV’ richness and radioactivity parameters. Non-parametric MANOVA analysis based on ASVs occurrence or abundances revealed that geographical location was the main factor influencing ASVs distribution. Interestingly, 238U was the second factor that explained diatom ASV structure. Among the ASVs in the mineral springs monitored, ASV associated with one of the genetic variants of Planothidium frequentissimum was well represented in the springs and with higher levels of 238U, suggesting its high tolerance to this particular radionuclide. This diatom species may therefore represent a bio-indicator of high natural levels of uranium.

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring.

Molecular diet analysis enables detection of diatom and cyanobacteria DNA in the gut of Macoma balthica.

Detritivores are essential to nutrient cycling, but are often neglected in trophic networks, due to difficulties with determining their diet. DNA analysis of gut contents shows promise of trophic link discrimination, but many unknown factors limit its usefulness. For example, DNA can be rapidly broken down, especially by digestion processes, and DNA provides only a snapshot of the gut contents at a specific time. Few studies have been performed on the length of time that prey DNA can be detected in consumer guts, and none so far using benthic detritivores. Eutrophication, along with climate change, is altering the phytoplankton communities in aquatic ecosystems, on which benthic detritivores in aphotic soft sediments depend. Nutrient-poor cyanobacteria blooms are increasing in frequency, duration, and magnitude in many water bodies, while nutrient-rich diatom spring blooms are shrinking in duration and magnitude, creating potential changes in diet of benthic detritivores. We performed an experiment to identify the taxonomy and quantify the abundance of phytoplankton DNA fragments on bivalve gut contents, and how long these fragments can be detected after consumption in the Baltic Sea clam Macoma balthica. Two common species of phytoplankton (the cyanobacteria Nodularia spumigena or the diatom Skeletonema marinoi) were fed to M. balthica from two regions (from the northern and southern Stockholm archipelago). After removing the food source, M. balthica gut contents were sampled every 24 hours for seven days to determine the number of 23S rRNA phytoplankton DNA copies and when the phytoplankton DNA could no longer be detected by quantitative PCR. We found no differences in diatom 18S rRNA gene fragments of the clams by region, but the southern clams showed significantly more cyanobacteria 16S rRNA gene fragments in their guts than the northern clams. Interestingly, the cyanobacteria and diatom DNA fragments were still detectable by qPCR in the guts of M. balthica one week after removal from its food source. However, DNA metabarcoding of the 23S rRNA phytoplankton gene found in the clam guts showed that added food (i.e. N. spumigena and S. marinoi) did not make up a majority of the detected diet. Our results suggest that these detritivorous clams therefore do not react as quickly as previously thought to fresh organic matter inputs, with other phytoplankton than large diatoms and cyanobacteria constituting the majority of their diet. This experiment demonstrates the viability of using molecular methods to determine feeding of detritivores, but further studies investigating how prey DNA signals can change over time in benthic detritivores will be needed before this method can be widely applicable to both models of ecological functions and conservation policy.

Radiation exposure of microorganisms living in radioactive mineral springs

The TIRAMISU collaboration gathers expertise from biologists, physicists, radiochemists and geologists within the Zone-Atelier Territoires Uranifères (ZATU) in France to study the response of microorganisms living in naturally radioactive mineral springs. Mineral springs are isolated ecosystems, extremely stable over geological time scales, which display different physicochemical and radiological parameters compared to their surroundings. Water and sediment samples collected in 27 mineral springs of the volcanic Auvergne region (Massif Central, France) have been analyzed for their microbial biodiversity and radionuclide content. Among the microorganisms present, diatoms (a microalgae species), widely used as environmental bioindicators of water quality, have shown to display an exceptional abundance of teratogenic forms in the most radioactive springs studied (radon activity up to 3700 Bq/L). The current work presents a first assessment of the dose received by the diatoms inhabiting naturally radioactive mineral springs. The radiological risk for microorganisms living in freshwater environments was estimated using the ERICA tool. Most of the sampled mineral springs were highly above the risk threshold of 10 mGy/h due to the large concentrations of radium in the sediments (up to 50 Bq/g). The complete radiological data on water and sediments are used as inputs to Monte Carlo simulations at micro- (GATE) and nano- (Geant4-DNA) scale in order to assess the direct and indirect damages on the diatom DNA.

Long-term preservation of biomolecules in lake sediments: potential importance of physical shielding by recalcitrant cell walls.

Even though lake sediments are globally important organic carbon (OC) sinks, the controls on long-term OC storage in these sediments are unclear. Using a multiproxy approach, we investigate changes in diatom, green algae, and vascular plant biomolecules in sedimentary records from the past centuries across five temperate lakes with different trophic histories. Despite past increases in the input and burial of OC in sediments of eutrophic lakes, biomolecule quantities in sediments of all lakes are primarily controlled by postburial microbial degradation over the time scales studied. We, moreover, observe major differences in biomolecule degradation patterns across diatoms, green algae, and vascular plants. Degradation rates of labile diatom DNA exceed those of chemically more resistant diatom lipids, suggesting that chemical reactivity mainly controls diatom biomolecule degradation rates in the lakes studied. By contrast, degradation rates of green algal and vascular plant DNA are significantly lower than those of diatom DNA, and in a similar range as corresponding, much less reactive lipid biomarkers and structural macromolecules, including lignin. We propose that physical shielding by degradation-resistant cell wall components, such as algaenan in green algae and lignin in vascular plants, contributes to the long-term preservation of labile biomolecules in both groups and significantly influences the long-term burial of OC in lake sediments.

Characterizing temporal variability in streams supports nutrient indicator development using diatom and bacterial DNA metabarcoding

Interest in developing periphytic diatom and bacterial indicators of nutrient effects continues to grow in support of the assessment and management of stream ecosystems and their watersheds. However, temporal variability could confound relationships between indicators and nutrients, subsequently affecting assessment outcomes. To document how temporal variability affects measures of diatom and bacterial assemblages obtained from DNA metabarcoding, we conducted weekly periphyton and nutrient sampling from July to October 2016 in 25 streams in a 1293 km2 mixed land use watershed. Measures of both diatom and bacterial assemblages were strongly associated with the percent agriculture in upstream watersheds and total phosphorus (TP) and total nitrogen (TN) concentrations. Temporal variability in TP and TN concentrations increased with greater amounts of agriculture in watersheds, but overall diatom and bacterial assemblage variability within sites—measured as mean distance among samples to corresponding site centroids in ordination space—remained consistent. This consistency was due in part to offsets between decreasing variability in relative abundances of taxa typical of low nutrient conditions and increasing variability in those typical of high nutrient conditions as mean concentrations of TP and TN increased within sites. Weekly low and high nutrient diatom and bacterial metrics were more strongly correlated with site mean nutrient concentrations over the sampling period than with same day measurements and more strongly correlated with TP than with TN. Correlations with TP concentrations were consistently strong throughout the study except briefly following two major precipitation events. Following these events, biotic relationships with TP reestablished within one to three weeks. Collectively, these results can strengthen interpretations of survey results and inform monitoring strategies and decision making. These findings have direct applications for improving the use of diatoms and bacteria, and the use of DNA metabarcoding, in monitoring programs and stream site assessments.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom.

To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method. The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed. When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05). The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Bibliometrics Analysis in English and Chinese Literature on Drowning in Forensic Medicine from 1991 to 2020.

To explore the research hotspots and development trends of the field of forensic drowning from 1991 to 2020 by bibliometrics methods. Based on Web of Science, CNKI database, Wanfang Data knowledge service platform, python 3.9.2, CiteSpace 5.8.R3, Gephi 0.9.2, etc. were used to analyze the publishing trends, countries/regions, institutions, authors and topics of the study on drowning. A total of 631 English literature were obtained, including 59 articles from Chinese authors, and 386 Chinese literature were obtained. The Chinese and English journals with the largest number of related literatures were Chinese Journal of Forensic Science (80 articles) and Forensic Science International (106 articles), respectively. Japan published the most articles in English, and China ranked third. Osaka City Univ (Japan, 28 articles) published the most English articles, and Guangzhou Forens Sci Inst (China, 22 articles) ranked second. Among Chinese literature, Guangzhou Forens Sci Inst (32 articles) published the most. The topic analysis of Chinese and English literature showed that diatom examination, virtual autopsy, postmortem biochemical examination, the nature of death, and postmortem submersion interval were the hot spots of current research, but English literature had more studies on new technologies and methods, while Chinese literature was more inclined to practice, application and experience summary. The number of literature in forensic medicine on drowning is relatively stable. The scope of international and domestic collaborations in this field is still limited. The automated examination of diatoms, the establishment of diatom DNA barcodes and virtual autopsy will be the most important research hotspots in the coming period and are expected to achieve breakthroughs in drowning diagnosis, drowning location inference, postmortem submersion interval estimation, etc.

Radiation exposure of microorganisms living in radioactive mineral springs

The TIRAMISU collaboration gathers expertise from biologists, physicists, radiochemists and geologists within the Zone-Atelier Territoires Uranifères (ZATU) in France to analyze the radiation exposure of microorganisms living in naturally radioactive mineral springs. These springs are small waterbodies that are extremely stable over geological time scales and display different physicochemical and radiological parameters compared to their surroundings. Water and sediment samples collected in 27 mineral springs of the volcanic Auvergne region (Massif Central, France) have been studied for their microbial biodiversity and their radionuclide content. Among the microorganisms present, microalgae (diatoms), widely used as environmental indicators of water quality, have shown to display an exceptional abundance of teratogenic forms in the most radioactive springs studied (radon activity up to 3700 Bq/L). The current work presents a first assessment of the dose received by the diatoms inhabiting these ecosystems. According to ERICA tool, microorganisms living in most of the sampled mineral springs were exposed to dose rates above 10 μGy/h due to the large concentration of radium in the sediments (up to 50 Bq/g). Radiological analyses of water and sediments were used as inputs to Monte Carlo simulations at micro-(GATE) and nano- (Geant4-DNA) scale in order to assess the direct and indirect damages on the diatom DNA.

Blowin' in the wind: Dispersal, structure, and metacommunity dynamics of aeolian diatoms in the McMurdo Sound region, Antarctica.

Diatom metacommunities are structured by environmental, historical, and spatial factors that are often attributed to organism dispersal. In the McMurdo Sound region (MSR) of Antarctica, wind connects aquatic habitats through delivery of inorganic and organic matter. We evaluated the dispersal of diatoms in aeolian material and its relation to the regional diatom metacommunity using light microscopy and 18S rRNA high-throughput sequencing. The concentration of diatoms ranged from 0 to 8.76 * 106 valves · g-1 dry aeolian material. Up to 15% of whole cells contained visible protoplasm, indicating that up to 3.43 * 104 potentially viable individuals could be dispersed in a year to a single 2 -cm2 site. Diatom DNA and RNA was detected at each site, reinforcing the likelihood that we observed dispersal of viable diatoms. Of the 50 known morphospecies in the MSR, 72% were identified from aeolian material using microscopy. Aeolian community composition varied primarily by site. Meanwhile, each aeolian community was comprised of morphospecies found in aquatic communities from the same lake basin. These results suggest that aeolian diatom dispersal in the MSR is spatially structured, is predominantly local, and connects local aquatic habitats via a shared species pool. Nonetheless, aeolian community structure was distinct from that of aquatic communities, indicating that intrahabitat dispersal and environmental filtering also underlie diatom metacommunity dynamics. The present study confirms that a large number of diatoms are passively dispersed by wind across a landscape characterized by aeolian processes, integrating the regional flora and contributing to metacommunity structure and landscape connectivity.

Potential for cross-contamination of diatom DNA samples when using toothbrushes

The use of toothbrushes and similar devices for sampling diatoms from hard surfaces is a well-established approach. Toothbrushes are routinely cleaned and reused when sampling for analysis by light microscopy. This paper looks at the scale of contamination encountered when this technique is used to sample diatoms for metabarcoding analyses, as well as at the scale of contamination to be expected if stream, rather than distilled water, is used to wash diatoms from stones. Although some contamination attributable to toothbrushes was detected, read numbers were low and had no effect on index calculation or ecological status estimates. However, if the primary focus of a study is to thoroughly document diversity in a sample, then even this small level of contamination may be unacceptable and more stringent measures may be required.

Validation of river typologies using molecular information from diatoms

The European Water Framework Directive (2000/60/EC) includes biological assessment of water bodies that has been implemented for many years. Indicator organisms such as diatoms respond to geological and hydrological features of rivers by modifying their structure. Therefore, when implementing the WFD, it was necessary to establish type-specific reference conditions to be able to measure the deviations of sampled communities due to anthropogenic impact.HTS-related eDNA metabarcoding has been developed to complement or even replace traditional approaches for its rapid, low-cost and highly accurate identification of communities for assessment of rivers’ ecological status (e.g. Mortágua et al., 2019; Pérez-Burillo et al. 2020) and proved to provide even more in-depth information about biological elements. The use of this information without assignment to species is being addressed once it eliminates the limiting factor of the reference database incompleteness and may provide new ecological information (e.g. Feio et al., 2020; Rivera et al., 2020). Since WFD requires the establishment of reference conditions for each water body type, for eDNA methods’ implementation it will be essential to review, confirm or reformulate, and perhaps create new typologies. Hereupon, the aim of this study is to analyze diatom communities from different typologies of Portuguese rivers resulting from DNA metabarcoding data and compare it with current typology system. To do so, we will verify the consistency of biological groups included in each type, validate the molecular data, analyze the correspondence of OTU/ISU/ESV to environmental characteristics of rivers. A total of 154 sampling sites were selected from central Portugal and northern Portugal in 2017 and 2019. The biofilm was collected for morphological identification and DNA sequencing of diatoms. Reference sites were selected for 4 river types (mountain, littoral, small and medium-large northern rivers) based on a set of pressure information (water quality, hydromorphology, land use and riparian zones). Diatom inventories were obtained from molecular and morphological analysis. DNA sequences were treated using Mothur software which processed two bioinformatic strategies in order to obtain the final ISU and OTU tables, while ESVs were treated with DADA2 package from R. Identification and counting of diatom valves took place under the light microscope concerning the morphological approach. We expect results to validate the molecular data for each typology either when assigning to species or not, and to understand whether it is necessary to establish new typologies for future use of the molecular approach in ecological assessment of rivers. Directive, W. F. (2000). Water Framework Directive. Journal reference OJL, 327, 1-73. Feio, M. J., Serra, S. R., Mortágua, A., Bouchez, A., Rimet, F., Vasselon, V., &amp;amp; Almeida, S. F. P. (2020). A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms. Science of the Total Environment, 722, 137900. https://doi.org/10.1016/j.scitotenv.2020.137900 Mortágua, A., Vasselon, V., Oliveira, R., Elias, C., Chardon, C., Bouchez, A., ... &amp;amp; Almeida, S. F. P. (2019). Applicability of DNA metabarcoding approach in the bioassessment of Portuguese rivers using diatoms. Ecological indicators, 106, 105470. https://doi.org/10.1016/j.ecolind.2019.105470 Pérez-Burillo, J., Trobajo, R., Vasselon, V., Rimet, F., Bouchez, A., &amp;amp; Mann, D. G. (2020). Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers. Science of the Total Environment, 727, 138445. https://doi.org/10.1016/j.scitotenv.2020.138445 Rivera, S. F., Vasselon, V., Bouchez, A., &amp;amp; Rimet, F. (2020). Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software. Ecological indicators, 109, 105775. https://doi.org/10.1016/j.ecolind.2019.105775

The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardisation

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

Inside Front Cover: Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death

Inside Front Cover: Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death

Comparative study on diatom morphology and molecular identification in drowning cases

In the field of criminal investigations, in the event that a body is found in water, the ability to differentiate whether the cause of death was drowning or the body was murdered then dumped into water elsewhere is difficult but important for case detection. Detecting diatoms in human organs can be used to effectively identify if the cause of death was drowning. At present, diatom detection methods are roughly divided into morphological and molecular detection methods, but both methods have different limitations. In this study, a total of 79 samples from 23 victims in 19 known drowning deaths were collected. The diatom morphological identification combined with DNA metabarcoding technology was used to compare the reliability of the diatom detection method. Microscopic observations revealed that the positive detection rate of diatoms was 52.6 %, 26.3 % and 58.8 % respectively in the kidney, liver and lung samples. DNA metabarcoding analysis found that the positive detection rate of diatoms was 31.6 %, 31.6 % and 35.3 % respectively in kidney, liver and lung samples. When compared with barcode BacirbcL, barcode 18S605 detected more diatoms, while diatoms in BacirbcL were more consistent with environmental samples. The comparative analysis found that microscopic observations were not highly correlated with the identification results of DNA barcoding technology. There were no obvious differences in the effect of internal organs on diatom enrichment, and different organs should be tested at the same time. At present, the DNA barcode reference sequence is gravely insufficient and has many errors, which leads to restrictions in the application of this technology, resulting in many OTU not being accurately identified. This explains why the success rate of molecular identification is not higher than that of microscopic identification. Construction of a reliable diatom DNA barcode reference sequence database is an urgent task for drowning forensics.

Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death.

Diatoms are unicellular microalgae with cell wall made up of rigid silica found in all open water bodies. They thus resist degradation and hence are important tool to diagnose cause of death in drowned bodies. The nitric acid digestion method practiced conventionally in forensic science laboratories has limitations due to manual error. Plant chloroplast genes found in diatoms such as ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL-3P) and rbcL, universal plastidic amplicon (UPA), and photosynthesis I P700 apoprotein chlorophyll Al (psaA), which play an important role in photosystems I and II of photosynthesis, are tested to diagnose drowning in experimental mice. It was seen that psaA-2 showed amplification at 150bp in all biological samples. The sequences of psaA-2 gene marker showed 100% proximity to Thalassiosira weissflogii and rbcL-3P showed 99% resemblance to Pseudo-nitzschia multiseries. On the other hand, in postmortem drowned biological samples, the chloroplast-based gene marker failed to show any amplification.