Kimiko Uchii

Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.

Droplet Digital Polymerase Chain Reaction (PCR) Outperforms Real-Time PCR in the Detection of Environmental DNA from an Invasive Fish Species

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

Detection of cyprinid herpesvirus 3 DNA in river water during and after an outbreak.

The disease caused by cyprinid herpesvirus 3 (CyHV-3) brings catastrophic damages to cultivated carp and koi and to natural carp populations; however, the dynamics of the virus in environmental waters are unclear. In July 2007, CyHV-3 DNA was detected in a dead common carp collected from the Yura River in Kyoto Prefecture, Japan, and this was followed by mass mortality. We collected water samples at eight sites along the Yura River for 3 months immediately after confirmation of the disease outbreak and attempted to detect and quantify CyHV-3 DNA in the water samples using molecular biological methods. The virus concentration was carried out by the cation-coated filter method, while the purification of DNA from the samples was achieved using phenol-chloroform extraction and a commercial DNA extraction kit. CyHV-3 was detected by PCR using six sets of conditions, three sets of primers (SphI-5, AP, and B22Rh exon 1), and two volumes of template DNA, and was quantified using real-time PCR. Our results indicate broader distribution of CyHV-3, even though dead fish were found only in a limited area; moreover, the virus was present at high levels in the river not only during the mass mortality caused by the disease but also for at least 3 months after the end of mass mortality. Our results suggest the possibility of infection by CyHV-3 via environmental water. The sequences of CyHV-3 collected from the Yura River matched perfectly with that of the CyHV-3 Japanese strain, suggesting that they share the same origin.

Genetic and Physiological Characterization of the Intestinal Bacterial Microbiota of Bluegill (Lepomis macrochirus) with Three Different Feeding Habits

Bluegill (Lepomis macrochirus) in Lake Biwa, Japan, feed on benthic invertebrates (benthivorous type), aquatic plants (herbivorous type), and zooplankton (planktivorous type). To evaluate the effect of food on intestinal bacterial microbiota, we characterized and compared the intestinal microbiota of these three types of bluegill in terms of community-level physiological profile (CLPP) and genetic structure. The CLPP was analyzed using Biolog MicroPlates (Biolog, Inc., Hayward, CA, USA), and multivariate analysis of variance revealed that the CLPP of intestinal microbiota differed significantly between any pairs of the three types of bluegill. The genetic profiles were analyzed by temperature gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments, and multidimensional scaling indicated the existence of specific intestinal bacterial structures for both the benthivorous and the planktivorous types. These results suggest that the hosts feeding habit can be one factor controlling the intestinal microbiota of fish in the natural environment.

Transmission dynamics of an emerging infectious disease in wildlife through host reproductive cycles.

Emerging infectious diseases are major threats to wildlife populations. To enhance our understanding of the dynamics of these diseases, we investigated how host reproductive behavior and seasonal temperature variation drive transmission of infections among wild hosts, using the model system of cyprinid herpesvirus 3 (CyHV-3) disease in common carp. Our main findings were as follows: (1) a seroprevalence survey showed that CyHV-3 infection occurred mostly in adult hosts, (2) a quantitative assay for CyHV-3 in a host population demonstrated that CyHV-3 was most abundant in the spring when host reproduction occurred and water temperature increased simultaneously and (3) an analysis of the dynamics of CyHV-3 in water revealed that CyHV-3 concentration increased markedly in breeding habitats during host group mating. These results indicate that breeding habitats can become hot spots for transmission of infectious diseases if hosts aggregate for mating and the activation of pathogens occurs during the host breeding season.

A peculiar relationship between genetic diversity and adaptability in invasive exotic species: bluegill sunfish as a model species

A peculiar relationship exists between population genetics and invasion biology. Introduced populations often suffer a depletion of genetic variation, but they can persist and adapt to new environments. Here, we show that this relationship is observed in bluegill sunfish (Lepomis macrochirus), an invasive exotic fish in Japan. Genetic analysis using selectively neutral genetic markers reconfirmed that the bluegill introduced into Japan from the United States in 1960 had a single origin with only 15 founders. The analysis also shows that in the process of range expansion, the introduced bluegills experienced severe depletion of genetic diversity due to the founder effect and/or genetic drift. Despite such a decline in genetic diversity, the bluegill populations exhibited a divergent feeding morphology in response to the colonized environments. Such a morphological divergence can facilitate prey exploitation, thereby causing a greater negative impact on native prey resources. Further, in a trophically polymorphic bluegill population in Lake Biwa, physiological characteristics and genetic structures of the intestinal bacterial communities were associated with the difference in diet among the trophic morphs in the host bluegill population. This empirical evidence suggests that despite the severe decline in genetic diversity, the introduced bluegill populations rapidly adapted to the new environment and formed diverse functional relationships with the native bacterial community. Thus, these findings suggest that genetic variation at selectively neutral markers does not always predict adaptability and invasiveness in introduced populations.

Quantification of cyprinid herpesvirus 3 in environmental water by using an external standard virus.

ABSTRACT Cyprinid herpesvirus 3 (CyHV-3), a lethal DNA virus that spreads in natural lakes and rivers, infects common carp and koi. We established a quantification method for CyHV-3 that includes a viral concentration method and quantitative PCR combined with an external standard virus. Viral concentration methods were compared using the cation-coated filter and ultrafiltration methods. The recovery of virus-like particles was similar for the two methods (cation-coated filter method, 44% ± 19%, n = 3; ultrafiltration method, 50% ± 3%, n = 3); however, the former method was faster and more suitable for routine determinations. The recovery of seeded CyHV-3 based on the cation-coated filter method varied by more than 3 orders of magnitude among the water samples. The recovery yield of CyHV-3 was significantly correlated with that of the seeded λ phage, and the average ratio of λ to the CyHV-3 recovery yield was 1.4, indicating that λ is useful as an external standard virus for determining the recovery yield of CyHV-3. Therefore, to quantify CyHV-3 in environmental water, a known amount of λ was added as an external standard virus to each water sample. Using this method, CyHV-3 DNA was detected in 6 of the 10 (60%) types of environmental water tested; the highest concentration of CyHV-3 DNA was 2 × 105 copies liter−1. The lowest recovery limit of CyHV-3 DNA was 60 copies liter−1. This method is practical for monitoring CyHV-3 abundance in environmental water.

A novel environmental DNA approach to quantify the cryptic invasion of non-native genotypes

The invasion of non‐native species that are closely related to native species can lead to competitive elimination of the native species and/or genomic extinction through hybridization. Such invasions often become serious before they are detected, posing unprecedented threats to biodiversity. A Japanese native strain of common carp (Cyprinus carpio) has become endangered owing to the invasion of non‐native strains introduced from the Eurasian continent. Here, we propose a rapid environmental DNA‐based approach to quantitatively monitor the invasion of non‐native genotypes. Using this system, we developed a method to quantify the relative proportion of native and non‐native DNA based on a single‐nucleotide polymorphism using cycling probe technology in real‐time PCR. The efficiency of this method was confirmed in aquarium experiments, where the quantified proportion of native and non‐native DNA in the water was well correlated to the biomass ratio of native and non‐native genotypes. This method provided quantitative estimates for the proportion of native and non‐native DNA in natural rivers and reservoirs, which allowed us to estimate the degree of invasion of non‐native genotypes without catching and analysing individual fish. Our approach would dramatically facilitate the process of quantitatively monitoring the invasion of non‐native conspecifics in aquatic ecosystems, thus revealing a promising method for risk assessment and management in biodiversity conservation.

Trophic polymorphism in bluegill sunfish (Lepomis macrochirus) introduced into Lake Biwa: evidence from stable isotope analysis

Trophic polymorphism was recently reported in introduced bluegill (Lepomis macrochirus) in Lake Biwa, Japan, where three morphs are specialized in benthic invertebrates (benthivorous type), submerged aquatic plants (herbivorous type), and zooplankton (planktivorous type). We evaluated the long-term effects of food resource utilization by these trophic morphs using stable isotope ratios, δ15N and δ13C. A significant difference in δ15N was found between the benthivorous and planktivorous types. The planktivorous type had the higher δ15N value, which corresponded with the value expected from its prey, zooplankton. The lower δ15N value of the benthivorous type would be derived from the lower δ15N values of benthic prey organisms compared to zooplankton. These results support previous findings that the benthivorous and planktivorous types have different food resource utilization. In contrast, the δ15N and δ13C values of the herbivorous type were distinctly different from the expected values, indicating that this type was unlikely to utilize aquatic plants substantially, contradicting the results of the dietary analysis.

Nationwide Cyprinid herpesvirus 3 contamination in natural rivers of Japan.

Cyprinid herpesvirus 3 (CyHV-3) disease is a significant threat for common and koi carp cultivators and for freshwater ecosystems. To determine the prevalence of CyHV-3 in Japanese rivers, a nationwide survey of all national class-A rivers was undertaken in the Summer of 2008. The virus was concentrated from river water samples using the cation-coated filter method. CyHV-3 DNA was detected in 90 rivers, representing 90% of 103 successfully analysed rivers. More than 100,000 copies of CyHV-3 DNA per litre of sample were detected in four rivers, higher than that reported during the Yura River outbreak in 2007. For CyHV-3-positive rivers, the log CyHV-3 density was negatively correlated with the water temperature on the sampling date and positively correlated with the suspended solids and dissolved oxygen, which are annually averaged for each river. Our results demonstrate that virus detection using molecular biology techniques is a powerful tool for monitoring the presence of CyHV-3 in natural environments.