Impact of Host Genetics on Caecal Microbiota Composition and on Salmonella Carriage in Chicken

Abstract Background Salmonella Enteritidis (SE) is one of the major causes of human foodborne intoxication through the consumption of contaminated poultry products. Genetic selection of animals more resistant to Salmonella carriage and the modulation of gut microbiota are two promising ways of decreasing individual Salmonella carriage. This study aims to identify the main genetic and microbial factors controlling the individual levels of Salmonella carriage in chickens (Gallus gallus) in controlled experimental conditions. Two-hundred and forty animals from the White Leghorn inbred lines, N and 61, were infected by SE at 7 days of age. After infection, animals were kept in isolators to reduce the recontamination of birds by Salmonella. Caecal contents were sampled at 12 days post-infection and used for DNA extraction. Microbiota DNA was used to measure individual counts of SE by digital PCR and to determine the bacterial taxonomic composition through a 16S rRNA gene high-throughput sequencing approach. Results Results confirmed that the N line is more resistant to Salmonella carriage than the 61 line, and that intra-line variability is higher for the 61 line. Furthermore, the 16S analysis showed strong significant differences in microbiota taxonomic composition between the two lines. Out of 617 Operational Taxonomic Units (OTUs), over 390 were differentially abundant between the two lines. Furthermore, within the 61 line, we found a difference in the microbiota taxonomic composition between high and low Salmonella carriers, with 39 differentially abundant OTUs. Finally, via metagenome functional prediction based on 16S data, we identified several metabolic pathways potentially associated to microbiota taxonomic differences (e.g. butyrate metabolism) between high and low carriers. Conclusions Overall, this study demonstrates that the caecal microbiota composition of the N and 61 lines is influenced by the host genetics, which could be one of the reasons why these lines differ for their Salmonella carriage in experimental infection conditions.

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Vaccination against the brown stomach worm, Teladorsagia circumcincta, followed by parasite challenge, induces inconsistent modifications in gut microbiota composition of lambs

Parasites & Vectors ◽

10.1186/s13071-021-04688-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

James Rooney ◽

Alba Cortés ◽

Riccardo Scotti ◽

Daniel R. G. Price ◽

Yvonne Bartley ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sequence Data ◽

Rrna Gene ◽

Helminth Parasites ◽

Microbiota Composition ◽

Related Factors ◽

Experimental Conditions ◽

Teladorsagia Circumcincta ◽

Host Parasite

Abstract Background Growing evidence points towards a role of gastrointestinal (GI) helminth parasites of ruminants in modifying the composition of the host gut flora, with likely repercussions on the pathophysiology of worm infection and disease, and on animal growth and productivity. However, a thorough understanding of the mechanisms governing helminth-microbiota interactions and of their impact on host health and welfare relies on reproducibility and replicability of findings. To this aim, in this study, we analysed quantitative and qualitative fluctuations in the faecal microbiota composition of lambs vaccinated against, and experimentally infected with, the parasitic GI nematode Teladorsagia circumcincta over the course of two separate trials performed over two consecutive years. Methods Two trials were conducted under similar experimental conditions in 2017 and 2018, respectively. In each trial, lambs were randomly assigned to one of the following experimental groups: (i) vaccinated/infected, (ii) unvaccinated/infected and (iii) unvaccinated/uninfected. Faecal samples collected from individual animals were subjected to DNA extraction followed by high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene and bioinformatics and biostatistical analyses of sequence data. Results Substantial differences in the populations of bacteria affected by immunisation against and infection by T. circumcincta were detected when comparing data from the two trials. Nevertheless, the abundance of Prevotella spp. was significantly linked to helminth infection in both trials. Conclusions Despite the largely conflicting findings between the two trials, our data revealed that selected gut microbial populations are consistently affected by T. circumcincta infection and/or vaccination. Nevertheless, our study calls for caution when interpreting data generated from in vivo helminth-microbiome interaction studies that may be influenced by several intrinsic and extrinsic host-, parasite- and environment-related factors.

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Synchronized shift of oral, faecal and urinary microbiotas in bats and natural infection dynamics during seasonal reproduction

Royal Society Open Science ◽

10.1098/rsos.180041 ◽

2018 ◽

Vol 5 (5) ◽

pp. 180041 ◽

Cited By ~ 9

Author(s):

Muriel Dietrich ◽

Teresa Kearney ◽

Ernest C. J. Seamark ◽

Janusz T. Paweska ◽

Wanda Markotter

Keyword(s):

Disease Ecology ◽

High Throughput Sequencing ◽

Natural Infection ◽

Pathogen Transmission ◽

Comparative Approach ◽

Seasonal Reproduction ◽

Rrna Gene ◽

Microbiota Composition ◽

Infection Dynamics ◽

Bacterial Microbiota

Seasonal reproduction is a period of extreme physiological and behavioural changes, yet we know little about how it may affect host microbial communities (i.e. microbiota) and pathogen transmission. Here, we investigated shifts of the bacterial microbiota in saliva, urine and faeces during the seasonal reproduction of bats in South Africa, and test for an interaction in shedding patterns of both bacterial ( Leptospira ) and viral (adeno- and herpesviruses) agents. Based on a comparative approach in two cave-dwelling bat species and high-throughput sequencing of the 16S rRNA gene, we demonstrated a clear signature in microbiota changes over the reproduction season, consistent across the multiple body habitats investigated, and associated with the sex, age and reproductive condition of bats. We observed in parallel highly dynamic shedding patterns for both bacteria and viruses, but did not find a significant association between viral shedding and bacterial microbiota composition. Indeed, only Leptospira shedding was associated with alterations in both the diversity and composition of the urinary microbiota. These results illustrate how seasonal reproduction in bats substantially affects microbiota composition and infection dynamics, and have broad implications for the understanding of disease ecology in important reservoir hosts, such as bats.

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Seasonal dynamics in taxonomy and function within bacterial and viral metagenomic assemblages recovered from a freshwater agricultural pond

Environmental Microbiome ◽

10.1186/s40793-020-00365-8 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Jessica Chopyk ◽

Daniel J. Nasko ◽

Sarah Allard ◽

Anthony Bui ◽

Mihai Pop ◽

...

Keyword(s):

High Throughput Sequencing ◽

Antibiotic Resistance Genes ◽

Pond Water ◽

Taxonomic Composition ◽

Storm Event ◽

Rrna Gene ◽

Illumina Hiseq ◽

Culture Independent ◽

Freshwater Habitats ◽

And Function

Abstract Background Ponds are important freshwater habitats that support both human and environmental activities. However, relative to their larger counterparts (e.g. rivers, lakes), ponds are understudied, especially with regard to their microbial communities. Our study aimed to fill this knowledge gap by using culture-independent, high-throughput sequencing to assess the dynamics, taxonomy, and functionality of bacterial and viral communities in a freshwater agricultural pond. Results Water samples (n = 14) were collected from a Mid-Atlantic agricultural pond between June 2017 and May 2018 and filtered sequentially through 1 and 0.2 μm filter membranes. Total DNA was then extracted from each filter, pooled, and subjected to 16S rRNA gene and shotgun sequencing on the Illumina HiSeq 2500 platform. Additionally, on eight occasions water filtrates were processed for viral metagenomes (viromes) using chemical concentration and then shotgun sequenced. A ubiquitous freshwater phylum, Proteobacteria was abundant at all sampling dates throughout the year. However, environmental characteristics appeared to drive the structure of the community. For instance, the abundance of Cyanobacteria (e.g. Nostoc) increased with rising water temperatures, while a storm event appeared to trigger an increase in overall bacterial diversity, as well as the relative abundance of Bacteroidetes. This event was also associated with an increase in the number of antibiotic resistance genes. The viral fractions were dominated by dsDNA of the order Caudovirales, namely Siphoviridae and Myovirdae. Conclusions Overall, this study provides one of the largest datasets on pond water microbial ecology to date, revealing seasonal trends in the microbial taxonomic composition and functional potential.

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Assessment of the bacterial community structure in shallow and deep sediments of the Perdido Fold Belt region in the Gulf of Mexico

PeerJ ◽

10.7717/peerj.5583 ◽

2018 ◽

Vol 6 ◽

pp. e5583 ◽

Cited By ~ 9

Author(s):

Ma. Fernanda Sánchez-Soto Jiménez ◽

Daniel Cerqueda-García ◽

Jorge L. Montero-Muñoz ◽

Ma. Leopoldina Aguirre-Macedo ◽

José Q. García-Maldonado

Keyword(s):

Community Structure ◽

Bacterial Community ◽

Gulf Of Mexico ◽

Microbial Communities ◽

Bacterial Community Structure ◽

High Throughput Sequencing ◽

Hydrocarbon Contamination ◽

Rrna Gene ◽

Fold Belt ◽

Abundant Otus

The Mexican region of the Perdido Fold Belt (PFB), in northwestern Gulf of Mexico (GoM), is a geological province with important oil reservoirs that will be subjected to forthcoming oil exploration and extraction activities. To date, little is known about the native microbial communities of this region, and how these change relative to water depth. In this study we assessed the bacterial community structure of surficial sediments by high-throughput sequencing of the 16S rRNA gene at 11 sites in the PFB, along a water column depth gradient from 20 to 3,700 m, including five shallow (20–600 m) and six deep (2,800–3,700 m) samples. The results indicated that OTUs richness and diversity were higher for shallow sites (OTUs = 2,888.2 ± 567.88;H′ = 9.6 ± 0.85) than for deep sites (OTUs = 1,884.7 ± 464.2;H′ = 7.74 ± 1.02). Nonmetric multidimensional scaling (NMDS) ordination revealed that shallow microbial communities grouped separately from deep samples. Additionally, the shallow sites plotted further from each other on the NMDS whereas samples from the deeper sites (abyssal plains) plotted much more closely to each other. These differences were related to depth, redox potential, sulfur concentration, and grain size (lime and clay), based on the environmental variables fitted with the axis of the NMDS ordination. In addition, differential abundance analysis identified 147 OTUs with significant fold changes among the zones (107 from shallow and 40 from deep sites), which constituted 10 to 40% of the total relative abundances of the microbial communities. The most abundant OTUs with significant fold changes in shallow samples corresponded toKordiimonadales, Rhodospirillales,Desulfobacterales(Desulfococcus), Syntrophobacterales and Nitrospirales(GOUTA 19,BD2-6,LCP-6), whilstChromatiales,Oceanospirillales(Amphritea,Alcanivorax),Methylococcales,Flavobacteriales,Alteromonadales(Shewanella,ZD0117) andRhodobacteraleswere the better represented taxa in deep samples. Several of the OTUs detected in both deep and shallow sites have been previously related to hydrocarbons consumption. Thus, this metabolism seems to be well represented in the studied sites, and it could abate future hydrocarbon contamination in this ecosystem. The results presented herein, along with biological and physicochemical data, constitute an available reference for further monitoring of the bacterial communities in this economically important region in the GoM.

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Caecal Microbiota Composition of Experimental Laying Hens Differs According to Genetic Line and Vaccination to IBV

10.21203/rs.3.rs-177864/v1 ◽

2021 ◽

Author(s):

Marion Borey ◽

Bertrand Bed'Hom ◽

Nicolas Bruneau ◽

Jordi Estellé ◽

Frederik Larsen ◽

...

Keyword(s):

T Cells ◽

Gut Microbiota ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Microbiota Composition ◽

Genetic Line ◽

Vaccine Response ◽

Main Components ◽

Main Association ◽

Caecal Microbiota

Abstract BackgroundInteractions between the gut microbiota and the immune system may be involved in the vaccine response. In the present study, we studied the correlations between caecal microbiota composition and the immune response in six experimental laying hen lines harboring different haplotypes at the Major Histocompatibility complex (MHC), 7 weeks after their first vaccination against the infectious bronchitis virus (IBV). Two lines were previously considered as high responders (HR) to IBV vaccination and two other ones as low responders (LR). We explored to what extent the gut microbiota could be related to this variability through the characterization of caecal bacterial communities with a 16S rRNA gene amplicon sequencing approach, one week after an IBV infectious challenge. ResultsWe observed significant effects of both the vaccination and the genetic line on the microbiota, with lower bacterial richness in vaccinated chickens, especially in the Ruminococcaceae family. We also observed dissimilar caecal communities among the different MHC lines, and among the vaccinated and non-vaccinated animals. The effect of vaccination was similar in all the lines, with a reduced abundance of OTUs from the Ruminococcacea UCG-014 and Faecalibacterium genera, and an increased abundance of OTUs from the Eisenbergiella genus. The main association between the microbiota and the immune phenotypes implied TCRϒδ expression on TCRϒδ+ T cells, which especially shared negative associations with OTUs from the Escherichia-Shigella genus. Finally, opposing the HR and LR groups, we identified 3 OTUs more abundant in the HR group, from the Tyzzeralla family, the Angelakisella genus, and to a lower extent the Flavonifractor family. Conversely, an OTU from the CAG-352 genus was more abundant in the LR group. ConclusionsWe assessed that the caecal microbiota composition is associated with the IBV vaccine response in laying chickens, and that TCRϒδ expression on TCRϒδ+ T cells appears as one of the main components potentially involved in this interaction.

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Murine Genetic Background Has a Stronger Impact on the Composition of the Gut Microbiota than Maternal Inoculation or Exposure to Unlike Exogenous Microbiota

Applied and Environmental Microbiology ◽

10.1128/aem.00826-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 9

Author(s):

Hila Korach-Rechtman ◽

Shay Freilich ◽

Shiran Gerassy-Vainberg ◽

Keren Buhnik-Rosenblau ◽

Yael Danin-Poleg ◽

...

Keyword(s):

Gut Microbiota ◽

Inbred Lines ◽

Mouse Genetics ◽

Rrna Gene ◽

Continuous Exposure ◽

Microbiota Composition ◽

Microbial Composition ◽

Cross Model ◽

Host Genetics ◽

Reciprocal Hybrids

ABSTRACT The gut microbiota is a complex ecosystem, affected by both environmental factors and host genetics. Here, we aim at uncovering the bacterial taxa whose gut persistence is controlled by host genetic variation. We used a murine model based on inbred lines BALB/c and C57BL/6J and their F1 reciprocal hybrids (♀C57BL/6J × ♂BALB/c; ♀BALB/c × ♂C57BL/6J). To guarantee genetic similarity of F1 offspring, including the sex chromosomes, we used only female mice. Based on 16S rRNA gene sequencing, we found that the genetically different inbred lines present different microbiota, whereas their genetically identical F1 reciprocal hybrids presented similar microbiota. Moreover, the F1 microbial composition differed from that of both parental lines. Twelve taxa were shown to have genetically controlled gut persistence, while none were found to show maternal effects. Nine of these taxa were dominantly inherited by the C57BL/6J line. Cohousing of the parental inbred lines resulted in a temporary and minor shift in microbiota composition, which returned back to the former microbial composition following separation, indicating that each line tends to maintain a unique bacterial signature reflecting the line. Taken together, our findings indicate that mouse genetics has an effect on the microbial composition in the gut, which is greater than maternal effect and continuous exposure to different microbiota of the alternative line. Uncovering the bacterial taxa associated with host genetics and understanding their role in the gut ecosystem could lead to the development of genetically oriented probiotic products, as part of the personalized medicine approach. IMPORTANCE The gut microbiota play important roles for their host. The link between host genetics and their microbial composition has received increasing interest. Using a unique reciprocal cross model, generating genetically similar F1 hybrids with different maternal inoculation, we demonstrate the inheritance of gut persistence of 12 bacterial taxa. No taxa identified as maternally transmitted. Moreover, cohabitation of two genetically different inbred lines did not dramatically affect the microbiota composition. Taken together, our results demonstrate the importance of the genetic effect over maternal inoculation or effect of exposure to unlike exogenous microbiota. These findings may lead to the development of personalized probiotic products, specifically designed according to the genetic makeup.

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Metataxonomic Review To Elucidate The Role of The Microbiome In Celiac Disease Across The Gastrointestinal Tract.

10.21203/rs.3.rs-794677/v1 ◽

2021 ◽

Author(s):

Juliana Arcila ◽

Viviana Loria-Kohen ◽

Ana Ramírez de Molina ◽

Enrique Carrillo de Santa Pau ◽

Laura Judith Judith Zambrano

Keyword(s):

Immune Response ◽

Celiac Disease ◽

Gastrointestinal Tract ◽

High Throughput Sequencing ◽

Short Chain Fatty Acids ◽

Taxonomic Composition ◽

Rrna Gene ◽

Immunogenic Peptides ◽

First Time

Abstract Background: Dysbiosis of the microbiome has been related to the Celiac disease (CeD) progress, an autoimmune disease characterised by gluten intolerance developed in genetically susceptible individuals under certain environmental factors. The microbiome contributes to CeD pathophysiology modulating the immune response by the action of short-chain fatty acids (SCFA), affecting gut barrier integrity allowing the entrance of gluten derived proteins, and degrading immunogenic peptides of gluten through endoprolyl peptidase enzymes. Results: We reviewed state of the art in taxonomic composition for CeD and compiled the larger dataset of 16S prokaryotic ribosomal RNA (rRNA) gene high-throughput sequencing for consensus profiling. We present for the first time an integrative analysis of metataxonomic data from CeD patients, including samples from different body sites (saliva, pharynx, duodenum, and stool). We found the presence of coordinated changes through the gastrointestinal tract characterised by an increase in Actinobacteria species in the upper tract (pharynx and duodenum), and an increase in Proteobacteria in the lower tract (duodenum and stool), as well as site-specific changes evidencing a dysbiosis in CeD patients' microbiota. Moreover, we described the effect of adherence to a gluten-free diet (GFD) evidenced by an increase in beneficial bacteria and a decrease in some Betaproteobacteriales but not fully restoring CeD-related dysbiosis. Conclusions: We illustrate that the gut microbiota acts as an enhancer of immune response in CeD through the production of lipopolysaccharides and other bacterial components that activate the immune response and by decrease SCFA producers bacteria. Furthermore, microbial changes observed through the gastrointestinal tract of CeD patients may help manage the disease and follow-up GFD treatment.

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Impact of proanthocyanidin-rich apple intake on gut microbiota composition and polyphenol metabolomic activity in healthy mildly hypercholesterolemic subjects

Proceedings of The Nutrition Society ◽

10.1017/s002966512000436x ◽

2020 ◽

Vol 79 (OCE2) ◽

Author(s):

Athanasios Koutsos ◽

Maria M. Ulaszewska ◽

Kajetan Trošt ◽

Jan Stanstrup ◽

Letizia Mariani ◽

...

Keyword(s):

Gut Microbiota ◽

Phenolic Acids ◽

High Throughput Sequencing ◽

Daily Intake ◽

Rrna Gene ◽

Microbiota Composition ◽

Microbial Metabolites ◽

Potential Health ◽

Hypercholesterolemic Subjects ◽

Gut Microbiota Composition

AbstractApples are a rich source of polyphenols and fiber. Proanthocyanidins (PAs), the largest polyphenolic class in apples, can reach the colon almost intact where they interact with the gut microbiota producing simple phenolic acids. These metabolites have the potential to modulate gut microbiota composition and activity and impact on host physiology. A randomized, controlled, crossover, dietary intervention study was performed to determine the broad effects of whole apple intake on fecal gut microbiota composition and activity. Forty heathy mildly hypercholesterolemic volunteers (23 women, 17 men), with a mean BMI (± SD) 25.3 ± 3.7 kg/m2 and age 51 ± 11 years, consumed 2 apples/day (Renetta Canada, rich in PAs), or a sugar matched control apple beverage, for 8 weeks separated by a 4-week washout period in a random order. Fecal and 24-h urine samples were collected before and after each treatment. The broad effects of apple intake on fecal gut microbiota composition were explored by the high throughput sequencing (HTS) of 16S rRNA gene lllumina MiSeq sequencing (V3-V4 region). Sequencing data analysis was performed using the Quantitative Insight Into Microbial Ecology (QIIME) open-source pipeline version 1.9.1. Specific bacterial groups were also enumerated using the quantitative Fluorescence In Situ Hybridization (FISH). Furthermore, the potential formation of microbial polyphenol metabolites, after apple intake, was explored in urine using Liquid Chromatography (LC) High-Resolution Mass Spectrometry (HRMS) metabolomics. Preliminary analysis showed no changes in gut microbiota abundances measured by Illumina MiSeq, after correction for multiple testing. Apple intake significantly decreased Enterobacteriaceae population (P = 0.04) compared to the control beverage, as determined with FISH. Twenty-four polyphenol microbial metabolites were identified in higher concentrations in the apple group (P < 0.05) compared to the control, including valerolactones, valeric and phenolic acids. In conclusion, preliminary data suggest that the daily intake of 2 Renetta Canada apples significantly decreased Enterobacteriaceae population, a family known for its pathogenic members, in healthy mildly hypercholesterolemic subjects. Moreover, several polyphenol microbial metabolites were identified, suggesting that microbial activity is crucial and a prerequisite for the absorption of apple polyphenols, producing active metabolites with potential health benefits.

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Metataxonomic review to elucidate the role of the microbiome in Celiac disease across the gastrointestinal tract

10.1101/2021.08.08.455560 ◽

2021 ◽

Author(s):

Juliana Estefania Arcila Galvis ◽

Viviana Loria-Kohen ◽

Ana Ramirez de Molina ◽

Enrique Castillo de Santa Pau ◽

Laura Judith Marcos-Zambrano

Keyword(s):

Immune Response ◽

Celiac Disease ◽

Gastrointestinal Tract ◽

High Throughput Sequencing ◽

Short Chain Fatty Acids ◽

Taxonomic Composition ◽

Rrna Gene ◽

Immunogenic Peptides ◽

First Time

Dysbiosis of the microbiome has been related to the Celiac disease (CeD) progress, an autoimmune disease characterised by gluten intolerance developed in genetically susceptible individuals under certain environmental factors. The microbiome contributes to CeD pathophysiology modulating the immune response by the action of short-chain fatty acids (SCFA), affecting gut barrier integrity allowing the entrance of gluten derived proteins, and degrading immunogenic peptides of gluten through endoprolyl peptidase enzymes. We reviewed state of the art in taxonomic composition for CeD and compiled the larger dataset of 16S prokaryotic ribosomal RNA (rRNA) gene high-throughput sequencing for consensus profiling. We present for the first time an integrative analysis of metataxonomic data from CeD patients, including samples from different body sites (saliva, pharynx, duodenum, and stool). We found the presence of coordinated changes through the gastrointestinal tract characterised by an increase in Actinobacteria species in the upper tract (pharynx and duodenum), and an increase in Proteobacteria in the lower tract (duodenum and stool), as well as site-specific changes evidencing a dysbiosis in CeD patients' microbiota. Moreover, we described the effect of adherence to a gluten-free diet (GFD) evidenced by an increase in beneficial bacteria and a decrease in some Betaproteobacteriales but not fully restoring CeD-related dysbiosis. We illustrate that the gut microbiota acts as an enhancer of immune response in CeD through the production of lipopolysaccharides and other bacterial components that activate the immune response and by decrease SCFA producers bacteria. Furthermore, microbial changes observed through the gastrointestinal tract of CeD patients may help manage the disease and follow-up GFD treatment.

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Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Scientific Reports ◽

10.1038/s41598-021-83318-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatsuhiko Hoshino ◽

Ryohei Nakao ◽

Hideyuki Doi ◽

Toshifumi Minamoto

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Correlation Coefficients ◽

Environmental Dna ◽

Digital Pcr ◽

Absolute Quantification ◽

Taqman Probe ◽

Individual Species ◽

Natural Environments ◽

Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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