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The use of environmental DNA (eDNA) is becoming prevalent as a novel method of ecological monitoring. Although eDNA can provide critical information on the distribution and biomass of particular taxa, the DNA sequences of an organism remain unaltered throughout its existence, which complicates the accurate identification of crucial events, including spawning. Therefore, we examined DNA methylation as a novel source of information from eDNA, considering that the methylation patterns in eggs and sperm released during spawning differ from those of somatic tissues. Despite its potential applications, little is known about eDNA methylation, including its stability and methods for detection and quantification. Therefore, we conducted tank experiments and performed methylation analysis targeting 18S rDNA through bisulphite amplicon sequencing. In the target region, eDNA methylation was not affected by degradation and was equivalent to the methylation rate of genomic DNA from somatic tissues. Unmethylated DNA, abundant in the ovaries, was detected in the eDNA released during fish spawning. These results indicate that eDNA methylation is a stable signal reflecting targeted gene methylation and further demonstrate that germ cell-specific methylation patterns can be used as markers for detecting fish spawning.
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Transmission of antimicrobial-resistant bacteria among humans, animals, and the environment is a growing concern worldwide. The distribution of an international high-risk fluoroquinolone-resistant Escherichia coli clone, ST131, has been documented in clinical settings. However, the transmission of ST131 from humans to surrounding environments remains poorly elucidated. To comprehend the current situation and identify the source of ST131 in nature, we analyzed the genetic features of ST131 isolates from the aquatic environment (lake/river water) and wildlife (fox, raccoon, raccoon dog, and deer) and compared them with the features of isolates from humans in Japan using accessory and core genome single nucleotide polymorphism (SNP) analyses. We identified ST131 isolates belonging to the same phylotype and genome clusters (four of eight clusters were concomitant) with low SNP distance between the human isolates and those from the aquatic environment and wildlife. These findings warn of ST131 transmission between humans and the surrounding environment in Japan.
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Periodic monitoring can provide important information for the protection of endangered fish, sustainable use of fishery resources and management of alien species. Previous studies have attempted to monitor fish using non-invasive environmental DNA (eDNA) technology, generally employing quantitative PCR to quantify the eDNA concentration. However, the throughput was limited. High-throughput metabarcoding technology can detect the DNA of multiple species simultaneously in a single experiment but does not provide sufficient quantification. In this study, we applied a quantitative metabarcoding approach to simultaneously quantify the eDNA concentration of an entire fish assemblage in a small reservoir over two summer seasons. Traditional surveys were also conducted to investigate the individuals of fish. The eDNA concentrations were quantified using quantitative metabarcoding, and the fish species detected using this approach were highly consistent with the results of traditional fish monitoring. A significant positive relationship was observed between the eDNA concentration and fish species abundance. Seasonal changes in fish community structure were estimated using eDNA concentrations, which may reveal the activity seasons of different fish. The eDNA concentrations of different fish species peaked at different water temperatures, reflecting the differential responses of fish species to this environmental factor. Finally, by detecting outlier eDNA concentrations, the spawning activities of 13 fish species were estimated, 12 of which were roughly consistent with the current knowledge of fish spawning periods. These results indicate that quantitative eDNA metabarcoding with dozens of sampling times is useful for the simultaneous ecological monitoring of multiple fish species.
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DNA Ambiental , Animais , DNA Ambiental/genética , Biodiversidade , Estações do Ano , Código de Barras de DNA Taxonômico/métodos , Monitoramento Ambiental/métodos , Peixes/genética , EcossistemaRESUMO
BACKGROUND: Mycetoma is a chronic disease affecting the skin and subcutaneous tissue endemic in the tropical and subtropical regions. Several bacteria and fungi can cause mycetoma, but fungal mycetoma (eumycetoma) is challenging because the treatment requires a combination of a long-term antifungal agent and surgery. Although the transmission route has not yet been elucidated, infection from the soil is a leading hypothesis. However, there are few soil investigation studies, and the geographical distribution of mycetoma pathogens is not well documented. Here, we used multiplex real-time PCR technology to identify three fungal species from soil samples. METHODS: In total, 64 DNA samples were extracted from soil collected in seven villages in an endemic area in Sennar State, Sudan, in 2019. Primers and fluorescent probes specifically targeting the ribosomal DNA of Madurella mycetomatis, Falciformispora senegalensis, and F. tompkinsii were designed. RESULTS: Multiplex real-time PCR was performed and identified the major pathogen, M. mycetomatis that existed in most sites (95%). In addition, two other pathogens were identified from some sites. This is the first report on the use of this technique for identifying the eumycetoma causative microorganisms. CONCLUSIONS: This study demonstrated that soil DNA investigation can elucidate the risk area of mycetoma-causative agents. The results will contribute to the design of prevention measures, and further large-scale studies may be effective in understanding the natural habitats of mycetoma pathogens.
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Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-ß. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.
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Ciona intestinalis , Anfioxos , Animais , Humanos , Globinas/genética , Ciona intestinalis/genética , Anfioxos/genética , Vertebrados/genética , Sequência de Aminoácidos , Família Multigênica , Filogenia , Evolução MolecularRESUMO
Environmental DNA (eDNA) is an increasingly popular tool in biological and ecological studies. As a biproduct of its increasing use, large number of eDNA samples are being collected and stored, that potentially contain information of many non-target species. One potential use for these eDNA samples is a surveillance and early detection of pathogens and parasites that are otherwise difficult to detect. Echinococcus multilocularis is such a parasite with serious zoonotic concern, and whose range has been expanding. If eDNA samples from various studies can be repurposed in detecting the parasite, it can significantly reduce the costs and efforts in surveillance and early detection of the parasite. We designed and tested a new set of primer-probe for detecting E. multilocularis mitochondrial DNA in environmental medium. Using this primer-probe set, we conducted real-time PCR on repurposed eDNA samples collected from three streams flowing through an area of Japan endemic to the parasite. We detected the DNA of E. multilocularis in one of the 128 samples (0.78%). The discovery suggests that while detecting E. multilocularis using eDNA samples is possible, the rate of detection appear to be very low. However, given the naturally low prevalence of the parasite among wild hosts in endemic areas, the repurposed eDNAs may still be a valid option for surveillance in newly introduced areas with the reduced cost and efforts. Further work is needed to assess and improve the effectiveness of using eDNA for detection of E. multilocularis.
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DNA Ambiental , Equinococose , Echinococcus multilocularis , Animais , Echinococcus multilocularis/genética , Equinococose/veterinária , DNA Ambiental/genética , Reação em Cadeia da Polimerase em Tempo Real , ÁguaRESUMO
Loss of biodiversity is a serious concern, and amphibians are particularly threatened. Most small salamanders in Japan are endangered. Distributional information is fundamental to the conservation of these rare species; however, small salamanders are generally difficult to locate or catch. Environmental DNA analysis is an effective survey method for monitoring such rare species. The conventional polymerase chain reaction (PCR) method, which combines PCR amplification with subsequent electrophoresis, and the real-time PCR method, which uses fluorescent material, are commonly used for this purpose. In this study, a comparison of these two detection methods was conducted using a rare salamander species, Hynobius boulengeri, as a model case. We compared three points: (i) detection sensitivity, (ii) influence of environmental factors related to detection, and (iii) time and financial costs of the two methods. To perform this comparison, we developed a real-time PCR detection assay, conducted field surveys, and compared the time and financial costs of conventional and real-time PCR methods. The comparison showed no statistical difference in the detection sensitivity from field samples, and the effects of environmental factors tended to be similar. In addition, the financial cost was lower for the conventional PCR method while the time cost was lower for the real-time PCR method. Therefore, selecting eDNA detection methods based on objectives, time, and financial costs will promote efficient monitoring and contribute to the conservation of rare species.
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DNA Ambiental , Urodelos , Animais , Urodelos/genética , DNA Ambiental/genética , Reação em Cadeia da Polimerase em Tempo Real , Japão , Monitoramento Ambiental/métodosRESUMO
In recent years, environmental DNA (eDNA) technology has been used in a variety of water environments. Environmental DNA concentrations in marine samples tend to be lower than those in freshwater samples, and few studies have explored methods to improve the recovery yields of eDNA from seawater samples. In this study, we compared different seawater preservation solutions (RNAlater or ATL) to improve eDNA yields. The eDNA concentrations of vertebrate and invertebrate species were compared using species-specific eDNA assays, and the number of detected fish and their compositions were compared using metabarcoding analysis. ATL treatment resulted in significantly higher eDNA yields for both vertebrate and invertebrate species than RNAlater treatment. Metabarcoding analysis revealed non-significant effects of preservation on the number of detected species and species composition. These results suggest that ATL treatment improves DNA yields without changing the species composition compared with the commonly used RNAlater treatment. The findings of this study will reduce false-negative outcomes and provide highly reliable results in future biological surveys.
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DNA Ambiental , Animais , DNA Ambiental/genética , Água do Mar , DNA/química , Peixes/genética , Água Doce , Monitoramento Ambiental/métodosRESUMO
Antimicrobial resistance is a global concern requiring a one-health approach. Given wild animals can harbor antimicrobial-resistant bacteria (ARB), we investigated their presence in 11 fecal samples from wild animals using deoxycholate hydrogen sulfide lactose agar with or without cefotaxime (CTX, 1 mg/L). Thus, we isolated CTX-resistant Escherichia coli from two Japanese red fox fecal samples. One strain was O83:H42-ST1485-fimH58 CTX-M-55-producing E. coli carrying the genes aph(3â³)-Ib, aph(3')-Ia, aph(6)-Id, mdf(A), sitABCD, sul2, tet(A), and tet(B), whereas the other was O25:H4-ST131-fimH30 CTX-M-14-producing E. coli carrying mdf(A) and sitABCD and showing fluoroquinolone resistance. Thus, the presence of extended-spectrum ß-lactamase producers in wild foxes suggests a spillover of ARB from human activities to these wild animals.
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Infecções por Escherichia coli , Escherichia coli , Animais , Humanos , Escherichia coli/genética , Raposas , beta-Lactamases/genética , Japão , Antagonistas de Receptores de Angiotensina , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Inibidores da Enzima Conversora de Angiotensina , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/microbiologiaRESUMO
Preventing mosquito-borne infectious diseases requires that vector mosquitoes are monitored and controlled. Targeting immature mosquitoes (eggs, larvae, and pupae), which have less mobility than adults, is an effective management approach. However, conducting these surveys is often difficult due to the limitations of morphological classification and survey costs. The application of environmental DNA (eDNA) analysis can solve these issues because it allows easy estimation of species distribution and morphology-independent species identification. Although a few previous studies have reported mosquito eDNA detection, there is a gap in knowledge regarding the dynamics related to the persistence of immature mosquito eDNA. We used Culex pipiens pallens, a vector of West Nile fever, as a model species. First, we developed a species-specific detection assay and confirmed its specificity using in silico and in vitro tests. Next, we conducted laboratory experiments using breeding tanks. Water samples were collected at each developmental stage. In addition, water samples were collected daily until the seventh day after emergence from the pupae. We quantified eDNA using real-time PCR with the developed assay to investigate the dynamics of mosquito eDNA. The specificity of the developed assay was confirmed by in silico and in vitro tests. Mosquito eDNA was detected at all developmental stages and detected up to seven days after emergence of pupae. In particular, high concentrations of eDNA were detected immediately after hatching from eggs and after emergence from pupae. Highly frequent positive eDNA signals were continuously detected between egg hatching and pupa hatching. Mosquito eDNA was detected immediately after the eggs were introduced, and eDNA-positive detections continued until pupae emergence, suggesting that eDNA analysis is useful for monitoring mosquito larvae. In the future, monitoring immature mosquitoes using eDNA analysis will contribute to prevent mosquito-borne infectious diseases.
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Doenças Transmissíveis , Culex , Culicidae , DNA Ambiental , Animais , Culex/genética , Culicidae/genética , Larva/genética , Mosquitos Vetores/genética , Pupa/genética , ÁguaRESUMO
Farmed anguillid eels are frequently stocked into natural fresh waters to enhance eel resources, but little is known about what happens to these eels or their interactions with wild eels after stocking. A recent study observed a depressed survival and growth rate of farmed Japanese eels when they were reared with wild eels, which indicated that wild eels might interfere with the survival and growth of farmed-and-stocked eels through intraspecific competition. To contribute to improving eel stocking efficiency, the growth of farmed-and-stocked Japanese eels was compared among four rivers with different wild eel densities using mark-and-recapture studies. Based on the 2-year recapture survey after stocking, it was found that the density of the farmed-and-stocked eels was not significantly different among rivers. The daily growth rates of farmed-and-stocked eels in the rivers with lower wild eel density were significantly higher than those of the eels stocked into the rivers with higher wild eel density. The farmed-and-stocked eels moved significantly greater distances downstream than wild eels that showed sedentary behaviour. This and previous studies indicate that significant questions remain about the effectiveness of stocking farmed eels into water bodies where naturally recruited wild eels are present.
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Anguilla , Animais , Água Doce , RiosRESUMO
In an era of severe biodiversity loss, biological monitoring is becoming increasingly essential. The analysis of environmental DNA (eDNA) has emerged as a new approach that could revolutionize the biological monitoring of aquatic ecosystems. Over the past decade, macro-organismal eDNA analysis has undergone significant developments and is rapidly becoming established as the golden standard for non-destructive and non-invasive biological monitoring. In this review, I summarize the development of macro-organismal eDNA analysis to date and the techniques used in this field. I also discuss the future perspective of these analytical methods in combination with sophisticated analytical techniques for DNA research developed in the fields of molecular biology and molecular genetics, including genomics, epigenomics, and single-cell technologies. eDNA analysis, which to date has been used primarily for determining the distribution of organisms, is expected to develop into a tool for elucidating the physiological state and behaviour of organisms. The fusion of microbiology and macrobiology through an amalgamation of these technologies is anticipated to lead to the future development of an integrated biology.
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DNA Ambiental , Biodiversidade , Código de Barras de DNA Taxonômico/métodos , Ecossistema , Monitoramento Ambiental/métodosRESUMO
Mycetoma is a tropical disease caused by several fungi and bacteria present in the soil. Fungal mycetoma and eumycetoma are especially challenging to treat; therefore, prevention, early diagnosis, and early treatment are important, but it is also necessary to understand the geographic distribution of these pathogenic fungi. In this study, we used DNA metabarcoding methodology to identify fungal species from soil samples. Soil sampling was implemented at seven villages in an endemic area of Sennar State in Sudan in 2019, and ten sampling sites were selected in each village according to land-use conditions. In total, 70 soil samples were collected from ground surfaces, and DNA in the soil was extracted with a combined method of alkaline DNA extraction and a commercial soil DNA extraction kit. The region for universal primers was selected to be the ribosomal internal transcribed spacer one region for metabarcoding. After the second PCR for DNA library preparation, the amplicon-based DNA analysis was performed using next-generation sequencing with two sets of universal primers. A total of twelve mycetoma-causative fungal species were identified, including the prime agent, Madurella mycetomatis, and additional pathogens, Falciformispora senegalensis and Falciformispora tompkinsii, in 53 soil samples. This study demonstrated that soil DNA metabarcoding can elucidate the presence of multiple mycetoma-causative fungi, which may contribute to accurate diagnosis for patient treatment and geographical mapping.
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Besouros , Madurella , Micetoma , Animais , DNA , Primers do DNA , DNA Fúngico/análise , DNA Fúngico/genética , Humanos , Madurella/genética , Micetoma/microbiologia , Solo , Sudão/epidemiologiaRESUMO
Environmental DNA (eDNA) can be a powerful tool for detecting the distribution and abundance of target species. This study aimed to test the longevity of eDNA in marine sediment through a tank experiment and to use this information to reconstruct past faunal occurrence. In the tank experiment, juvenile jack mackerel (Trachurus japonicus) were kept in flow-through tanks with marine sediment for two weeks. Water and sediment samples from the tanks were collected after the removal of fish. In the field trial, sediment cores were collected in Moune Bay, northeast Japan, where unusual blooms of jellyfish (Aurelia sp.) occurred after a tsunami. The samples were analyzed by layers to detect the eDNA of jellyfish. The tank experiment revealed that after fish were removed, eDNA was not present in the water the next day, or subsequently, whereas eDNA was detectable in the sediment for 12 months. In the sediment core samples, jellyfish eDNA was detected at high concentrations above the layer with the highest content of polycyclic aromatic hydrocarbons, reflecting tsunami-induced oil spills. Thus, marine sediment eDNA preserves a record of target species for at least one year and can be used to reconstruct past faunal occurrence.
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DNA Ambiental/genética , Perciformes/genética , Cifozoários/genética , Tsunamis , Animais , Monitoramento Ambiental/métodos , Peixes/genética , Sedimentos Geológicos , Preservação Biológica/métodosRESUMO
The spread of antimicrobial-resistant bacteria (ARB) in natural environments including wild animals is a concern for public health. Birds cover large areas, and some fly across borders to migrate in large flocks. As a migratory bird, the Greater White-fronted Goose (Anser albifrons) travels to Miyajimanuma, North Japan, each spring and autumn. To investigate the ARB in migratory birds and their surroundings, we collected 110 fecal samples of A. albifrons and 18 water samples from Miyajimanuma in spring and autumn of 2019. Isolation of Escherichia coli was performed using selective agars with or without antimicrobials (cefazolin and nalidixic acid). Isolates of E. coli were recovered from 56 fecal samples (50.9%) and five water samples (27.8%) on agars without antimicrobials. No isolates were recovered on agars with antimicrobials. One E. coli isolate derived from a fecal sample exhibited resistance to ß-lactams (ampicillin and cefazolin), whereas all other isolates exhibited susceptibility to all tested antimicrobials. The resistant isolate harbored blaACC, which could be transferred to other bacteria and confer resistance to ß-lactams. These results suggest a low prevalence of antimicrobial resistance in wild migratory birds and their living environments; however, wild migratory birds sometimes carry ARB harboring transferrable antimicrobial resistance genes and therefore present a risk of spreading antimicrobial resistance.
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Anti-Infecciosos , Escherichia coli , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Ecossistema , Gansos , Japão , Testes de Sensibilidade Microbiana/veterinária , PrevalênciaRESUMO
Alien ant species (Formicidae, Hymenoptera) cause serious damage worldwide. Early detection of invasion and rapid management are significant for controlling these species. However, these attempts are sometimes hindered by the need for direct detection techniques, such as capture, visual observation, or morphological identification. In this study, we demonstrated that environmental DNA (eDNA) analysis can be used as a monitoring tool for alien ants using Linepithema humile (Argentine ant), one of the most invasive ants, as a model species. We designed a new real-time PCR assay specific to L. humile and successfully detected eDNA from the surface soil. The reliability of eDNA analysis was substantiated by comparing eDNA detection results with traditional survey results. Additionally, we examined the relationship between eDNA concentration and distance from nests and trails. Our results support the effectiveness of eDNA for alien ant monitoring and suggest that this new method could improve our ability to detect invasive ant species.
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DNA Ambiental/isolamento & purificação , Monitoramento Ambiental , Solo/química , Animais , Formigas/química , Formigas/genética , DNA Ambiental/genética , Humanos , Espécies IntroduzidasRESUMO
A lack of reliable tools for determining the presence and distribution of fish species can impede understanding of predator-prey interactions and fishery management. Conventional fish survey methods are invasive, and can be size or species selective. Combining netting and electrofishing is a current method used to monitor fish species in Phayao Lake (Kwan Phayao), Thailand. However, the methods are inefficient and time-consuming. Recently, locals who rely on inland fisheries in Kwan Phayao expressed their deep concerns about the giant snakehead, Channa micropeltes (Cuvier, 1831) destroying other fish there. The giant snakehead prey on many commercially important fish species, as the prey species is reduced, negative effects on both biodiversity and the fishery sector could follow. Here, an eDNA-based survey was developed to detect the presence of the giant snakehead. Water samples were collected from six sites within Kwan Phayao and 17 sites in Ing River where water flowed into and out of Kwan Payao. The eDNA of the giant snakehead was detected in water samples from all collection sites using the developed qPCR assay with various concentrations. The eDNA was shown here to be a sensitive and reliable tool for fish surveillance so there will be a better chance for developing an effective management strategy.
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DNA Ambiental/genética , Monitoramento Ambiental/métodos , Peixes/genética , Animais , Biodiversidade , Código de Barras de DNA Taxonômico/métodos , DNA Ambiental/análise , DNA Ambiental/isolamento & purificação , Pesqueiros/tendências , Espécies Introduzidas , Lagos , Rios , TailândiaRESUMO
Understanding the processes of environmental DNA (eDNA) persistence and degradation is essential to determine the spatiotemporal scale of eDNA signals and accurately estimate species distribution. The effects of environmental factors on eDNA persistence have previously been examined; however, the influence of the physiochemical and molecular states of eDNA on its persistence is not completely understood. Here, we performed meta-analyses including 26 previously published papers on the estimation of first-order eDNA decay rate constants, and assessed the effects of filter pore size, DNA fragment size, target gene, and environmental conditions on eDNA decay rates. Almost all supported models included the interactions between the filter pore size and water temperature, between the target gene and water temperature, and between the target gene and water source, implying the influence of complex interactions between the eDNA state and environmental conditions on eDNA persistence. These findings were generally consistent with the results of a reanalysis of a previous tank experiment which measured the time-series changes in marine fish eDNA concentrations in multiple size fractions after fish removal. Our results suggest that the mechanism of eDNA persistence and degradation cannot be fully understood without knowing not only environmental factors but also cellular and molecular states of eDNA in water. Further verification of the relationship between eDNA state and persistence is required by obtaining more information on eDNA persistence in various experimental and environmental conditions, which will enhance our knowledge on eDNA persistence and support our findings.
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DNA Ambiental , Peixes/genética , Água/química , Animais , TemperaturaRESUMO
The 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.