scholarly journals Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

2019 ◽  
Author(s):  
Elena Valsecchi ◽  
Jonas Bylemans ◽  
Simon J. Goodman ◽  
Roberto Lombardi ◽  
Ian Carr ◽  
...  
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Marine Biodiversity ◽  
Primer Sets ◽  
Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

scholarly journals Scanning ferry routes: looking for eDNA traces of marine mammals and their preys

2021 ◽  
Vol 4 ◽  
Author(s):  
Elena Valsecchi
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Trophic Levels ◽  
Systematic Sampling ◽  
Marine Biodiversity ◽  
Tyrrhenian Sea ◽  
Night Time ◽  
Species Specific

Marine environmental DNA (eDNA) surveys are becoming a promising approach to monitor biodiversity status and its variation over time. However, monitoring offshore areas could be extremely costly when using dedicated vessels, beside the impossibility to sample simultaneously geographically distant (even if adjacent) areas. The unexplored possibility of availing on operating ferries as an opportunistic platform for eDNA sampling offers several advantages besides opening limitless opportunities for systematic surveys on marine biodiversity.We present the results of both metabarcoding and barcoding approaches obtained from the analysis of water samples collected on board of a ferry boat along a pilot Mediterranean route crossing the Pelagos Sanctuary for Mediterranean Marine Mammals. The recently described MarVer primer sets (12SrDNA and 16SrDNA regions), specifically designed for the simultaneous detection of marine mammals and other marine vertebrates, were employed. The High Throughput Sequencing (HTS) outcome showed that the markers successfully detected most trophic levels of vertebrate marine communities, and classes, including bony fish, rays, cetaceans and birds. Ferry-based sampling allow to collect sample at any time of the day, and we indeed found diel differences in both quantitative and qualitative distribution of read counts. For instances, we observed an increased abundance of lantern fish amplicons in night-time collect samples (50%), reflecting nocturnal migration through the water column. In general, the number of read counts was significantly higher in nocturnal samples. Such diel differences within our sample indirectly provides evidence of the efficiency of the eDNA approach to detect contemporary signals in the sampled environment. Similarly, cetaceans were detected in correspondence of visual sightings (when these occurred, supplementary samples were collected). Rare species, such as the monk seal, are difficult to be detected in metabarcoding surveys, thus we opted to side the screening of the ferry-samples with a panel of species-specific qPCR assays, which were able to detect DNA traces of the endangered pinniped in the Tuscany archipelago (Tyrrhenian Sea) long before visual observations witnessed its presence in the same area. The study demonstrates the feasibility of using commercial shipping as a platform for eDNA marine sampling without dedicated survey cruises. Commercial shipping routes have potential to act as regular systematic sampling transects which can contribute to evaluating and monitoring marine biodiversity.

scholarly journals eDNA metabarcoding outperforms traditional fisheries sampling and reveals fine-scale heterogeneity in a temperate freshwater lake

2020 ◽  
Author(s):  
Rebecca R Gehri ◽  
Wesley A. Larson ◽  
Kristen Gruenthal ◽  
Nicholas Sard ◽  
Yue Shi
Keyword(s):  
Community Composition ◽  
Lake Water ◽  
Water Samples ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Aquatic Systems ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Great Promise ◽  
Fish Diversity

AbstractUnderstanding biodiversity in aquatic systems is critical to ecological research and conservation efforts, but accurately measuring species richness using traditional methods can be challenging. Environmental DNA (eDNA) metabarcoding, which uses high-throughput sequencing and universal primers to amplify DNA from multiple species present in an environmental sample, has shown great promise for augmenting results from traditional sampling to characterize fish communities in aquatic systems. Few studies, however, have compared exhaustive traditional sampling with eDNA metabarcoding of corresponding water samples at a small spatial scale. We intensively sampled Boardman Lake (137 ha) in Michigan, USA from May to June in 2019 using gill and fyke nets and paired each net set with lake water samples collected in triplicate. We analyzed water samples using eDNA metabarcoding with 12S and 16S fish-specific primers and compared estimates of fish diversity among methods. In total, we set 60 nets and analyzed 180 1 L lake water samples. We captured a total of 12 fish species in our traditional gear and detected 40 taxa in the eDNA water samples, which included all the species observed in nets. The 12S and 16S assays detected a comparable number of taxa, but taxonomic resolution varied between the two genes. In our traditional gear, there was a clear difference in the species selectivity between the two net types, and there were several species commonly detected in the eDNA samples that were not captured in nets. Finally, we detected spatial heterogeneity in fish community composition across relatively small scales in Boardman Lake with eDNA metabarcoding, but not with traditional sampling. Our results demonstrated that eDNA metabarcoding was substantially more efficient than traditional gear for estimating community composition, highlighting the utility of eDNA metabarcoding for assessing species diversity and informing management and conservation.

scholarly journals Comparative Analysis of the Fecal Bacterial Microbiota of Wintering Whooper Swans (Cygnus Cygnus)

2021 ◽  
Vol 8 ◽  
Author(s):  
Wenxia Wang ◽  
Songlin Huang ◽  
Liangliang Yang ◽  
Guogang Zhang
Keyword(s):  
Intestinal Microbiota ◽  
Relative Abundance ◽  
High Throughput Sequencing ◽  
The Body ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Illumina Hiseq ◽  
Linear Discriminant ◽  
Bacterial Microbiota ◽  
And Control

There are many and diverse intestinal microbiota, and they are closely related to various physiological functions of the body. They directly participate in the host's food digestion, nutrient absorption, energy metabolism, immune response, and many other physiological activities and are also related to the occurrence of many diseases. The intestinal microbiota are extremely important for maintaining normal physical health. In order to explore the composition and differences of the intestinal microbiota of whooper swans in different wintering areas, we collected fecal samples of whooper swans in Sanmenxia, Henan, and Rongcheng, Shandong, and we used the Illumina HiSeq platform to perform high-throughput sequencing of bacterial 16S rRNA genes. Comparison between Sanmenxia and Rongcheng showed no significant differences in ACE, Chao 1, Simpson, and Shannon indices (p > 0.05). Beta diversity results showed significant differences in bacterial communities between two groups [analysis of similarity (ANOSIM): R = 0.80, p = 0.011]. Linear discriminant analysis effect size (LEfSe) analysis showed that at the phylum level, the relative abundance of Actinobacteria was significantly higher in Sanmenxia whooper swans than Rongcheng whooper swans. At the genus level, the amount of Psychrobacter and Carnobacterium in Sanmenxia was significantly higher in Rongcheng, while the relative abundance Catellicoccus and Lactobacillus was significantly higher in Rongcheng than in Sanmenxia. This study analyzed the composition, characteristics, and differences of the intestinal microbiota of the whooper swans in different wintering environments and provided theoretical support for further exploring the relationship between the intestinal microbiota of the whooper swans and the external environment. And it played an important role in the overwintering physiology and ecology, population management, and epidemic prevention and control of whooper swans.

scholarly journals The Purple Sea Urchin Strongylocentrotus purpuratus Demonstrates a Compartmentalization of Gut Bacterial Microbiota, Predictive Functional Attributes, and Taxonomic Co-Occurrence

2019 ◽  
Vol 7 (2) ◽  
pp. 35 ◽  
Author(s):  
Joseph Hakim ◽  
Julie Schram ◽  
Aaron Galloway ◽  
Casey Morrow ◽  
Michael Crowley ◽  
...  
Keyword(s):  
Sea Urchin ◽  
High Throughput Sequencing ◽  
Sea Urchins ◽  
Community Integration ◽  
Strongylocentrotus Purpuratus ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Tide Pool ◽  
Alpha And Beta Diversity ◽  
Taxonomic Organization

The sea urchin Strongylocentrotus purpuratus (order Camarodonta, family Strongylocentrotidae) can be found dominating low intertidal pool biomass on the southern coast of Oregon, USA. In this case study, three adult sea urchins were collected from their shared intertidal pool, and the bacteriome of their pharynx, gut tissue, and gut digesta, including their tide pool water and algae, was determined using targeted high-throughput sequencing (HTS) of the 16S rRNA genes and bioinformatics tools. Overall, the gut tissue demonstrated Arcobacter and Sulfurimonas (Epsilonproteobacteria) to be abundant, whereas the gut digesta was dominated by Psychromonas (Gammaproteobacteria), Propionigenium (Fusobacteria), and Flavobacteriales (Bacteroidetes). Alpha and beta diversity analyses indicated low species richness and distinct microbial communities comprising the gut tissue and digesta, while the pharynx tissue had higher richness, more closely resembling the water microbiota. Predicted functional profiles showed Kyoto Encyclopedia of Genes and Genomes (KEGG) Level-2 categories of energy metabolism, membrane transport, cell motility, and signal transduction in the gut tissue, and the gut digesta represented amino acid, carbohydrate, vitamin and cofactor metabolisms, and replication and repair. Co-occurrence network analysis showed the potential relationships and key taxa, such as the highly abundant Arcobacter and Propionigenium, influencing population patterns and taxonomic organization between the gut tissue and digesta. These results demonstrate a trend of microbial community integration, allocation, predicted metabolic roles, and taxonomic co-occurrence patterns in the S. purpuratus gut ecosystem.

scholarly journals Illumina-based sequencing analysis of pathogenic microorganisms in dental caries patients of different Chinese ethnic groups

2019 ◽  
Vol 47 (10) ◽  
pp. 5037-5047
Author(s):  
Chen Yun ◽  
Li Zhiyan ◽  
Zhao Chong ◽  
Liu Jing ◽  
Zhang Xin ◽  
...  
Keyword(s):  
Dental Caries ◽  
Ethnic Groups ◽  
High Throughput Sequencing ◽  
Community Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Control Group ◽  
Sequencing Analysis ◽  
Pathogenic Microorganisms ◽  
Effective Prevention

Objective To analyze the pathogenic community diversity of dental caries patients from Tu, Hui, Tibetan, and Han Chinese ethnic groups. Methods Forty saliva samples were collected from the following patients with dental caries: Tu from Huzhu County (n = 10), Hui from Ping’an County (n = 10), Han from Xining city (n = 10), and Tibetan from Yushu (n = 10). High-throughput sequencing of bacterial 16S rRNA genes (V3-V4) was performed using the Illumina MiSeq sequencing platform. Results Based on 97% similarity clustering, operational taxonomic units of Tu, Hui, Tibetan, and Han ethnic groups were 181, 210, 38, and 67, respectively. In Tu patients, 11 phyla, 19 classes, and 89 genera were identified, compared with 13 phyla, 21 classes, and 113 genera in Hui patients, two phyla, four classes, and 21 genera in Tibetan patients, five phyla, nine classes, and 34 genera in Han patients, and four phyla, five classes, and 12 genera from the control group. The main pathogens of dental caries included Veillonella, Aggregatibacter, Leptotrichia, Bacteroides, Granulicatella, Streptococcus, and Prevotella. Conclusion The pathogenic microorganisms of dental caries differ greatly among Tu, Hui, Tibetan, and Han ethnic groups. These findings provide a theoretical basis for the effective prevention and treatment of dental caries in different Chinese populations.

scholarly journals 454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Alejandro Gonzalez-Martinez ◽  
Alejandro Rodriguez-Sanchez ◽  
Belén Rodelas ◽  
Ben A. Abbas ◽  
Maria Victoria Martinez-Toledo ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Nitrogen Removal ◽  
Annealing Temperature ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Universal Primers ◽  
Anammox Bacteria ◽  
Universal Primer ◽  
Optimum Annealing Temperature ◽  
Autotrophic Nitrogen Removal

Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for theBacteriadomain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.

scholarly journals Community and Proteomic Analysis of Anaerobic Consortia Converting Tetramethylammonium to Methane

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-14
Author(s):  
Wei-Yu Chen ◽  
Lucia Kraková ◽  
Jer-Horng Wu ◽  
Domenico Pangallo ◽  
Lenka Jeszeová ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
High Throughput Sequencing ◽  
Molecular Techniques ◽  
Upflow Anaerobic Sludge Blanket ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Uasb Reactor ◽  
Anaerobic Sludge ◽  
Methane Formation ◽  
Proteomic Approach

Tetramethylammonium-degrading methanogenic consortia from a complete-mixing suspended sludge (CMSS) and an upflow anaerobic sludge blanket (UASB) reactors were studied using multiple PCR-based molecular techniques and shotgun proteomic approach. The prokaryotic 16S rRNA genes of the consortia were analyzed by quantitative PCR, high-throughput sequencing, and DGGE-cloning methods. The results showed that methanogenicarchaeawere highly predominant in both reactors but differed markedly according to community structure. Community and proteomic analysis revealed thatMethanomethylovoransandMethanosarcinawere the major players for the demethylation of methylated substrates and methane formation through the reduction pathway of methyl-S-CoM and possibly, acetyl-CoA synthase/decarbonylase-related pathways. Unlike high dominance of oneMethanomethylovoranspopulation in the CMSS reactor, diverse methylotrophicMethanosarcinaspecies inhabited in syntrophy-like association with hydrogenotrophicMethanobacteriumin the granular sludge of UASB reactor. The overall findings indicated the reactor-dependent community structures of quaternary amines degradation and provided microbial insight for the improved understanding of engineering application.

Investigation of microbial communities on reverse osmosis membranes used for process water production

2007 ◽  
Vol 55 (8-9) ◽  
pp. 181-190 ◽  
Author(s):  
L.A. Bereschenko ◽  
A.J.M. Stams ◽  
G.H.J. Heilig ◽  
G.J.W. Euverink ◽  
M.M. Nederlof ◽  
...  
Keyword(s):  
16S Rrna ◽  
Reverse Osmosis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Universal Primers ◽  
Feed Water ◽  
Rrna Gene ◽  
Membrane Pore ◽  
Phylogenetic Affiliation ◽  
Ro Membrane

In the present study, the diversity and the phylogenetic affiliation of bacteria in a biofouling layer on reverse osmosis (RO) membranes were determined. Fresh surface water was used as a feed in a membrane-based water purification process. Total DNA was extracted from attached cells from feed spacer, RO membrane and product spacer. Universal primers were used to amplify the bacterial 16S rRNA genes. The biofilm community was analysed by 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) and the phylogenetic affiliation was determined by sequence analyses of individual 16S rDNA clones. Using this approach, we found that five distinct bacterial genotypes (Sphingomonas, Beta proteobacterium, Flavobacterium, Nitrosomonas and Sphingobacterium) were dominant genera on surfaces of fouled RO membranes. Moreover, the finding that all five “key players” could be recovered from the cartridge filters of this RO system, which cartridge filters are positioned before the RO membrane, together with literature information where these bacteria are normally encountered, suggests that these microorganisms originate from the feed water rather than from the RO system itself, and represent the fresh water bacteria present in the feed water, despite the fact that the feed water passes an ultrafiltration (UF) membrane (pore size approximately 40 nm), which is able to remove microorganisms to a large extent.

Sign in / Sign up

Export Citation Format

Share Document