scholarly journals Ichthyoplankton metabarcoding as a tool for studying fish reproductive dynamics

2021 ◽  
Vol 4 ◽  
Author(s):  
Daniel Teixeira ◽  
Heron Hilário ◽  
Gustavo Rosa ◽  
Guilherme Santos ◽  
Gilmar Santos ◽  
...  
Keyword(s):  
Fish Assemblages ◽  
Fish Larvae ◽  
Reference Sequence ◽  
12S Rrna ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Fish Eggs ◽  
Migration Routes ◽  
Dna Metabarcoding ◽  
Primer Sets

The study of ichthyoplankton composition, abundance and distribution is paramount to understand the reproductive dynamics of local fish assemblages. The analysis of these parameters allows the identification of spawning sites, nursery areas and migration routes. However, due to the lack of characters in early life stages, the morphological identification of ichthyoplankton is often impractical and many studies identify only fish larvae. Additionally, its accuracy shows great variation between taxonomists and laboratories according to their experience and specialty. DNA barcoding emerged as an alternative to provide assertive identification of fish eggs and larvae, but it becomes too expensive and laborious when the study demands the processing of huge amounts of organisms. DNA metabarcoding can overcome these limitations as a rapid, cost-effective, broad and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. Here, we present the identification of a sample containing 68 fish eggs and another containing 293 fish larvae from a single site in the São Francisco River Basin, Eastern Brazil, through DNA metabarcoding. We used a low-cost saline DNA extraction followed by PCR amplification with three primer sets targeting the 12S rRNA gene: MiFish (~170bp), Teleo_1 (~60bp), and NeoFish (~190bp). The latter was recently developed by our research group specifically for the identification of Neotropical fishes. All the amplified samples were sequenced in a single multiplexed Illumina MiniSeq run. We performed the filtering steps and assigned Amplicon Sequence Variants (ASVs) using a DADA2/Phyloseq based pipeline and a custom 12S reference sequence database including 101 species and 70 genera from the Jequitinhonha and São Francisco basins. The species Cyphocharax gilbert, Leporinus taeniatus, Megaleporinus elongatus, Prochilodus argenteus, P. costatus and Psalidodon fasciatus were detected by all three primer sets in the larva pool, while Pterygoplichthys etentaculatus was detected solely by NeoFish (Fig. 1). Within the egg pool, all three markers detected the species Characidium zebra, Curimatella lepidura, M. elongatus, Pimelodus fur and P. costatus, but Brycon orthotaenia was detected only by NeoFish, P. maculatus only by Teleo, and P. pohli by MiFish and Teleo (Fig. 1). The consistency in species detection among all three markers underpins the credibility of this method to accurately describe the sample composition. Considering that most of species were exclusive to the larvae or egg pool, our experiment highlights the importance of including the identification of fish eggs in reproduction studies, as it can provide additional information about which species are spawning in an area. Furthermore, the application of DNA metabarcoding to the study of ichthyoplankton can help decision makers create more informed guidelines for conservation of economically and ecologically important fish species.

scholarly journals Uncovering the Competences of Commonly Used Nutrient Media, Multi-primer Approach and Reference Databases in the Identification of Microalgae Diversity from Environmental Samples

2021 ◽  
Author(s):  
Amal A. Badr ◽  
Walid M. Fouad
Keyword(s):  
Molecular Level ◽  
The Other ◽  
Reference Database ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Genus Level ◽  
Reference Databases ◽  
Primer Sets ◽  
Abundance And Diversity ◽  
Level Identification

Abstract Microalgae are highly diverse microorganisms and have a variety of benefits and use across different fields. On the other hand, their overgrowth can be extremely dangerous to our environment, thus, making it particularly important to continuously manage and track their abundance and diversity to oversee any potential of extinction or overgrowth. The vast diversity of microalgae imposes the challenge of their identification through the most common and economical identification method, morphological identification, and the more recent molecular-level identification tools. To enhance the identification of microalgae, we targeted enrichment of total microalgae diversity present in an environmental sample using four different enrichment media (BG-11, BBM, Modified media (MM), and half-strength Murashige and Skoog medium (MS)). Morphological identification of the enriched microalgae diversity was conducted every 4-days to monitor the population dynamics. After 14-day the DNA was extracted from the enriched population for molecular-level identification using 16S rRNA gene regions V1-V3 and V4-V5 and 18 rRNA gene V4 region. To further enhance microalgae identification through molecular-level identification, we evaluated three reference databases (SILVA, Greengenes, and Protist Ribosomal Reference (PR2)) to reveal their competence in microalgae diversity identification. A total of 38 microalgae were identified morphologically to the genus level, and the highest number of microalgae were identified through MM media (36), followed by BG-11 and BBM, 31 and 26, respectively. While sequencing the three-primer sets using the three databases, 87 microalgae were identified to the genus level. The highest diversity was identified using the MM media (71 genera) followed by BG-11 (69 genera), BBM (67 genera). Our multiple-media, primer, and reference database approach enabled us to identify a high microalgae diversity that would have been missed if a single approach was used over the other.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Strengths and weaknesses of DNA-based monitoring: Assessing macroinvertebrates in 18 Finnish streams with metabarcoding and morphology

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for assessment of stream ecosystems with macroinvertebrates. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We tested metabarcoding using 18 macroinvertebrate samples from Finland using four primer sets. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Morphological identification was performed to the taxonomic level at which identification was reliable following standardized protocols. 3) We identified over twice the number of taxa, with greater species-level resolution, using DNA metabarcoding than morphology-based identification. For each sample, we detected more taxa by metabarcoding than by previous morphological methods, and all four primer sets showed similarly good performance. There was a significant linear correlation between sequence abundance and the number of taxa in each sample, but the scatter was up to two orders of magnitude. Ecological status assessment indices calculated from morphological and DNA metabarcoding datasets were mostly similar, with a few exceptions. With the recent drop in sequencing costs per sample, both methods identification are currently equally expensive. 4) We used actual samples for monitoring to demonstrate that DNA metabarcoding can achieve similar results and better taxonomic resolution than current morphological identification methods. Metabarcoding has thus already become a viable and reliable invertebrate identification method for stream assessment. However, to unlock the full potential of DNA metabarcoding for ecosystem assessment key problems in current laboratory protocols and reference databases, specified in this work, will require further attention.

scholarly journals Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

2017 ◽  
Author(s):  
Vasco Elbrecht ◽  
Edith Vamos ◽  
Kristian Meissner ◽  
Jukka Aroviita ◽  
Florian Leese
Keyword(s):  
Large Scale ◽  
Monitoring Program ◽  
Ecological Status ◽  
Taxonomic Resolution ◽  
Great Promise ◽  
Morphological Identification ◽  
Full Potential ◽  
Stream Monitoring ◽  
Dna Metabarcoding ◽  
Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

scholarly journals Development of novel PCR primer sets for DNA metabarcoding of aquatic insects, and the discovery of some cryptic species

2021 ◽  
Author(s):  
Masaki Takenaka ◽  
Koki Yano ◽  
Tomoya Suzuki ◽  
Koji Tojo
Keyword(s):  
Cryptic Species ◽  
Dna Barcoding ◽  
Environmental Science ◽  
Pcr Primers ◽  
12S Rrna ◽  
Universal Primer ◽  
Data Registration ◽  
Dna Database ◽  
Dna Metabarcoding ◽  
Primer Sets

DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification by using standardized genetic region(s). It can be a powerful tool in various fields of biology such as for revealing the existence of cryptic species and/or rare species and in environmental science such as when monitoring river biota. Biodiversity reduction in recent times has become one of the most serious environmental issues on a worldwide scale. DNA barcoding techniques require the development of sets of universal PCR primers for DNA metabarcoding. We tried to develop universal primer sets for the DNA barcoding of all insect groups. In this study, we succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region. A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. Such resulting database enhancements will serve as a powerful tool for increasingly accurate assessment of biodiversity and genetic diversity.

scholarly journals Challenges in assessing the Neotropical fishes using DNA metabarcoding

2021 ◽  
Vol 4 ◽  
Author(s):  
Izabela Mendes ◽  
Heron Hilário ◽  
Daniel Teixeira ◽  
Daniel Cardoso de Carvalho
Keyword(s):  
River Basin ◽  
Fish Species ◽  
Species Level ◽  
Reference Sequence ◽  
Single Individual ◽  
Neotropical Fishes ◽  
Two Samples ◽  
Dna Metabarcoding ◽  
Primer Sets ◽  
Neotropical Ichthyofauna

Species richness is a metric of biodiversity usually used in fish community assessment for monitoring programs. This metric is often obtained using traditional fisheries methods that rely on capture of target organisms, resulting in underestimation of fish species. DNA metabarcoding has been recognized as a powerful noninvasive alternative tool for fish biomonitoring and management. Despite the increasing popularity of this method for the assessment of aquatic megadiverse ecosystems, its implementation for studying the highly diverse Neotropical ichthyofauna still presents some challenges. One of them is to devise what primer set could reliably amplify the DNA of all fish species from a megadiverse river basin and have enough resolution to identify them. In order to identify and overcome these drawbacks, we have investigated the efficiency of the metabarcoding approach on Neotropical fishes using a mock sample containing genomic DNA of 18 fish species from the Jequitinhonha River basin, Eastern Brazil. We compared three primer sets targeting the 12S rRNA gene: two universal and widely used markers for fish metabarcoding [MiFish (~170bp) and Teleo_1 (~60bp)], and NeoFish (~190bp), recently developed by our research group specifically for the identification of Neotropical fishes (Milan et al., 2020). Two samples amplified using three primers were sequenced in a single multiplexed Illumina MiniSeq run, using normalized and non-normalized pools. Bioinformatic analyses were performed using a DADA2/Phyloseq based pipeline to perform filtering steps and to assign Amplicon Sequence Variants (ASVs). We used a custom 12S reference sequence database that included 190 specimens representing 101 species and 70 genera from the Jequitinhonha and São Francisco river basins. A total of 187 ASVs were recovered: 79, 66 and 42 for NeoFish, MiFish and Teleo_1, respectively. ASVs of unexpected species were identified for both pools (Fig. 1), though each of these ASVs had an abundance of less than 50 copies. In addition, species of the Hoplias and Prochilodus genera could not be identified at the species level, due to identical sequences within each genus, possibly because of the insufficient variation within the 12S region recovered by these primers’ amplicons. Unexpectedly, although a single individual of each species was placed in the pools, more than one ASV was identified for some species, likely caused by PCR biases. Overall, all primer sets displayed similar taxonomic resolution for the DNA pools and recovered all species, except for NeoFish, which could not detect Steindachneridion amblyurum due to an incompatibility in the 3’ of the NeoFish forward primer and Teleo_1, which could not identify Steindachnerina elegans. These results highlight the need of reliable databases in order to enable the full assignment of ASVs and OTUs to species level, and the importance of calibrating the DNA metabarcoding approach with mock samples to identify weaknesses and pivotal steps prior to the application on large scale DNA based biodiversity evaluation, that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

scholarly journals Application of Environmental DNA (eDNA) Metabarcoding Method to Identify Threatened Sulawesi Mammal Based on 12S rRNA Gene

2021 ◽  
Vol 29 (1) ◽  
pp. 114-121
Author(s):  
Bambang Suryobroto ◽  
Ahmad Abdul Jabbar ◽  
Puji Rianti
Keyword(s):  
Species Identification ◽  
High Throughput Sequencing ◽  
Genetic Material ◽  
Environmental Dna ◽  
Reference Sequence ◽  
12S Rrna ◽  
Rrna Gene ◽  
Mammal Species ◽  
Terrestrial Mammals ◽  
Salt Licks

Species detection and identification is a crucial steps in biodiversity assessment. Traditional methods are often invasive and resource intensive. The number of studies demonstrating successful of eDNA metabarcoding approach in species identification has increased rapidly in recent years. Some of large terrestrial mammals have reportedly utilize natural salt licks as a source of minerals in the diet and its genetic material left in the environment can be used to identify species from this site. An eDNA metabarcoding protocol had been carried out to identify Sulawesi mammals from Adudu natural salt-licks, Nantu Wildlife Reserve, Gorontalo. Environmental DNA were extracted from water samples, Amplicon libraries were prepared by PCR amplification and Illumina MiSeq high throughput sequencing. Reads processing and taxonomic assignment carried out in two bioinformatics packages, PipeCraft-1.0 and OBITools-2.11. Two endangered Sulawesi mammals species had been identified, i.e. lowland anoa (Bubalus depressicornis) and babirusa (Babyrousa babyrussa). The accuracy of mammal species identification using eDNA metabarcoding is affected by rigorous experimental procedures, DNA marker reliability, and availability of reference sequence database.

scholarly journals Characterization of the 12S rRNA Gene Sequences of the Harvester Termite Anacanthotermes ochraceus (Blattodea: Hodotermitidae) and Its Role as A Bioindicator of Heavy Metal Accumulation Risks in Saudi Arabia

Insects ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 51
Author(s):  
Reem Alajmi ◽  
Rewaida Abdel-Gaber ◽  
Noura AlOtaibi
Keyword(s):  
Heavy Metals ◽  
Metal Accumulation ◽  
Heavy Metal Accumulation ◽  
12S Rrna ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Termite Species ◽  
12S Rrna Gene ◽  
The Mean ◽  
High Concentrations

Termites are social insects of economic importance that have a worldwide distribution. Identifying termite species has traditionally relied on morphometric characters. Recently, several mitochondrial genes have been used as genetic markers to determine the correlation between different species. Heavy metal accumulation causes serious health problems in humans and animals. Being involved in the food chain, insects are used as bioindicators of heavy metals. In the present study, 100 termite individuals of Anacanthotermes ochraceus were collected from two Saudi Arabian localities with different geoclimatic conditions (Riyadh and Taif). These individuals were subjected to morphological identification followed by molecular analysis using mitochondrial 12S rRNA gene sequence, thus confirming the morphological identification of A. ochraceus. Furthermore, a phylogenetic analysis was conducted to determine the genetic relationship between the acquired species and other termite species with sequences previously submitted in the GenBank database. Several heavy metals including Ca, Al, Mg, Zn, Fe, Cu, Mn, Ba, Cr, Co, Be, Ni, V, Pb, Cd, and Mo were measured in both collected termites and soil samples from both study sites. All examined samples (termite and soil) showed high concentrations of metals with different concentrations and ratios. Generally, most measured metals had a significantly high concentration in soil and termites at Taif, except for Ca, Cd, Co, Cr, Cu, Mg, and Ni showing significantly high concentrations at Riyadh. Furthermore, termites accumulated higher amounts of heavy metals than the soil at both locations. The mean concentrations of the measured metals in soil samples were found to be in the descending order Ca ˃ Al ˃ Mg ˃ Zn ˃ Fe ˃ Cu ˃ Mn ˃ Ba ˃ Cr ˃ Co ˃ Be ˃ Ni ˃ V ˃ Pb ˃ Cd ˃ Mo, while it was Ca ˃ Mg ˃ Al ˃ Fe ˃ Zn ˃ Cu ˃ Mn ˃ Be ˃ Ba ˃ Pb ˃ Cr ˃ V ˃ Ni ˃ Cd ˃ Mo ˃ Co in termite specimens. The mean concentrations of the studied metals were determined in the soil and termite specimens at both locations. In addition, the contamination factor, pollution load index (PLI) and degree of contamination were calculated for all studied metals in different samples, indicating that both studied sites were polluted. However, Taif showed a significantly higher degree of pollution. Thus, the accurate identification of economically important insects, such as termites, is of crucial importance to plan for appropriate control strategies. In addition, termites are a good bioindicator to study land pollution.

scholarly journals Development of mitochondrial 12S rRNA gene for identification of dog and rat in beef using multiplex PCR

2019 ◽  
Vol 44 (1) ◽  
pp. 10 ◽  
Author(s):  
M. Cahyadi ◽  
I. M. Taufik ◽  
A. Pramono ◽  
Z. H. Abdurrahman
Keyword(s):  
Multiplex Pcr ◽  
Sequence Data ◽  
Uv Light ◽  
Reference Sequence ◽  
12S Rrna ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
12S Rrna Gene ◽  
Mitochondrial 12S Rrna

The 12S rRNA gene is one of unique regions in mitochondrial genome usually used for phylogenetic studies and species identification. The objective of present study was to develop species specific primers from mitochondrial 12S rRNA gene for identification of dog and rat in beef by using multiplex PCR assay. Three primer pairs of mitochondrial 12S rRNA gene specific for bovine, dog and rat were designed and selected to evaluate their specificity and fidelity. Moreover, a total of twelve DNA samples extracted from meat tissue were also prepared to test those primers using simplex and multiplex PCR. The PCR products were then visualized using 2% of agarose gel under the UV light and three of them were sequenced. In addition, sequence data were analyzed using Clustal Omega software and BLAST. The result showed that simplex PCR assay successfully amplified DNA targets which are respectively indicated by 155 bp (bovine), 244 bp (dog), and 491 bp (rat) of DNA bands. Furthermore, DNA sample sequences were identically similar to reference sequence used in this study. Multiplex and simplex PCR analyses also indicated that these primer pairs specifically amplified DNA target for each species in the samples containing various species. The results suggested that designed primers in this study could be used to identify dog and rat in raw beef containing these species meat. Further experiment should be conducted using meat-processed products and commercial meat products as samples.

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