Detection of invasive freshwater fish species using environmental DNA survey

Invasive species are one of the most significant problem in freshwater ecosystems. Most common non-native freshwater species in Turkish freshwater fish fauna are Prussian Carp (Carassius gibelio), North African Catfish (Clarias gariepinus), Nile Tilapia (Oreochromis niloticus) and Topmouth Gudgeon (Pseudorasbora parva).Recent studies showed that environmental DNA could be used to detect target species inhabiting the ecosystem with higher precision and less effort compared to traditional field surveys. In this study, eDNA approach was used to investigate non-native freshwater fish species from fifteen different locations of Upper Sakarya Basin. eDNA was successfully extracted from the water samples of locations where the species were visually observed. Mean amplification rate of eDNA was calculated as 77.03%.This study is the first environmental DNA study used in detection of four of the most common invasive freshwater fish species. Results clearly indicating that eDNA surveys could be used as an important molecular tool to monitor invasive fish species in freshwater ecosystems.     

基于环境DNA宏条形码技术的北京地区鱼类多样性调查和外来鱼种入侵风险评估

[目的]调查北京地区鱼类多样性和群落分布及评估外来鱼种的入侵风险.[方法]选取北京地区水库、湖泊和河流3种水体类型共33个采样点,于2020年6月10—17日开展水生态监测,利用环境DNA宏条形码技术对各样点的鱼类多样性和群落结构进行监测和分析,对目前北京地区水生态系统中本地鱼种和外来鱼种进行分类汇总,并评估典型外来入...     

Mycobacterium marinum infections in humans and tracing of its possible environmental sources

The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium?marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M.?marinum presence in the aquarists' fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n?=?20), aquarium animals (n?=?8), and an aquarium environment (n?= 10). Isolate identification was carried out using 16S?rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M.?marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium?kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium?peregrinum . The presence of M.?marinum was proven in the aquarium environments of two patients. Although M.?marinum is described as being present in water, it was detected only in fish.     

Environmental DNA detection of aquatic invasive plants in lab mesocosm and natural field conditions

Aquatic invasive plant species cause negative impacts to economies and ecosystems worldwide. Traditional survey methods, while necessary, often do not result in timely detections of aquatic invaders, which can be cryptic, difficult to identify, and exhibit very rapid growth and reproduction rates. Environmental DNA (eDNA) is a relatively new method that has been used to detect multiple types of animals in freshwater and marine ecosystems through tissues naturally shed from the organism into the water column or sediment. While eDNA detection has proven highly effective in the detection of aquatic animals, we know less about the efficacy of eDNA as an effective surveillance tool for aquatic plants. To address this disparity, we designed mesocosm experiments with Elodea species to determine the ability to detect accumulation and degradation of the DNA signal for aquatic plants, followed by field surveillance of the highly invasive Hydrilla verticillata in freshwaters across several U.S. geographic regions. In both lab and field experiments, we designed a high sensitivity quantitative PCR assay to detect the aquatic plant species. In both experiments, plant eDNA detection was successful; we saw accumulation of DNA when plants were introduced to tanks and a decrease in DNA over time after plants were removed. We detected eDNA in the field in areas of known Hydrilla distribution. Employing eDNA detection for aquatic plants will strengthen efforts for early detection and rapid response of invaders in global freshwater ecosystems.     

Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

The use of environmental DNA of fishes as an efficient method of determining habitat connectivity

This study demonstrated the use of environmental DNA (eDNA) to determine habitat connectivity for migration of fishes between the sea and river. Environmental DNA is DNA fragments released by fishes in water, which can be used as a species-specific marker of the presence/absence of the target species. A year-round water sampling regime at 15 sites on the Yodo River, Japan, was conducted to determine whether three major man-made barriers on the river inhibited the migration of fishes using species-specific detection of DNA fragments from three target migrant species, temperate seabass, Lateolabrax japonicus, flathead grey mullet, Mugil cephalus, and ayu, Plecoglossus altivelis altivelis. The presence/absence of eDNA from target species was consistent with known patterns of species’ seasonal migration. The detection of the DNA of temperate seabass and flathead grey mullet at sites upstream of the dam closest to the river mouth indicated successful upstream migration of these species via a fish ladder bypassing the dam. On the other hand, DNA of these two species was not detected from the upstream side of the two remaining dams, which are not equipped with fish ladders. Ayu is the only species among the three target species with a land-locked population in Lake Biwa located at the headwater of Yodo River. Ayu DNA was detected at most of the sites in the freshwater area during the warm months; however, in the coldest month of February, eDNA was only detected in the uppermost site of Yodo River at the southern tip of Lake Biwa. The eDNA we detected at this site suggests that it was derived from juvenile ayu spending their winter months in the lake. These results suggest that the eDNA analysis presented here can accurately track the seasonal migration of fishes in a river, demonstrating its application as an indicator of habitat connectivity for fishes in association with man-made barriers in a river. The sampling of eDNA involves merely scooping a tank full of water; therefore, it is a simple, rapid, and cost-effective method for long-term monitoring of habitat connectivity associated with the construction of barriers in a river.     

Laboratory and field validation of a simple method for detecting four species of non‐native freshwater fish using eDNA

This paper presents the first phase in the development and validation of a simple and reliable environmental (e)DNA method using conventional PCR to detect four species of non‐native freshwater fish: pumpkinseed Lepomis gibbosus, sunbleak Leucaspius delineatus, fathead minnow Pimephales promelas and topmouth gudgeon Pseudorasbora parva. The efficacy of the approach was demonstrated in indoor tank (44 l) trials in which all four species were detected within 24 h. Validation was through two field trials, in which L. gibbosus was detected 6–12 h after its introduction into outdoor experimental ponds and P. parva was successfully detected in disused fish rearing ponds where the species was known to exist. Thus, the filtration of small (30 ml) volumes of pond water was sufficient to capture fish eDNA and the approach emphasised the importance of taking multiple water samples of sufficient spatial coverage for detecting species of random or patchy distribution.     

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

Organic matter reduces the amount of detectable environmental DNA in freshwater

Environmental DNA (eDNA) is used for monitoring the occurrence of freshwater organisms. Various studies show a relation between the amount of eDNA detected and target organism abundance, thus providing a potential proxy for reconstructing population densities. However, environmental factors such as water temperature and microbial activity are known to affect the amount of eDNA present as well. In this study, we use controlled aquarium experiments using Gammarus pulex L. (Amphipoda) to investigate the relationship between the amount of detectable eDNA through time, pH, and levels of organic material. We found eDNA to degrade faster when organic material was added to the aquarium water, but that pH had no significant effect. We infer that eDNA contained inside cells and mitochondria is extra resilient against degradation, though this may not reflect actual presence of target species. These results indicate that, although estimation of population density might be possible using eDNA, measured eDNA concentration could, in the future, be corrected for local environmental conditions in order to ensure accurate comparisons.     

Infection Characteristics of Clonorchis sinensis Metacercariae in Fish from Republic of Korea

The infection nature of Clonorchis sinensis metacercariae (CsMc) in freshwater fish hosts is closely related to the transmission of human clonorchiasis. This article reviewed the infection characteristics of CsMc in freshwater fish in the Republic of Korea (Korea). The status of CsMc infection was examined in a total of 17,792 cyprinid fish of 49 species in 9 water systems, which included Hantan-/Imjin-gang, Han-gang, Geum-gang, Mangyeong-gang, Yeongsan-gang, Tamjin-gang, Seomjin-gang, Nakdong-gang, and streams in the east coastal areas from 2010 to 2020. The infection status of CsMc was examined according to cyprinid fish species and water systems, after which analyzed by endemicity and susceptibility index. The high endemicity was shown in the cyprinid fish from 3 regions (6.1%) in the upper reaches of Nakdong-gang, such as Banbyeon-cheon (stream), Yongjeon-cheon, and Wi-cheon. The moderate levels were observed in fishes from 8 regions (16.3%), and low endemicity was shown in fishes from 20 regions (40.8%). No CsMc were detected in fish from 18 regions (36.7%). The susceptibility of CsMc in index fish, Puntungia herzi, was found to be a reliable index without examination of other fish species. CsMc infection rates were closely related to subfamily groups in the cyprinid fish hosts in a highly endemic area. In Korea, a total of 58 fish species in 10 families has been listed as the second intermediate hosts for C. sinensis. This review provides several novel features of CsMc infection and clarifies the species of second intermediate freshwater fish host in Korea.     

Next‐generation monitoring of aquatic biodiversity using environmental DNA metabarcoding

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90–0.99) vs. 0.58 (CI = 0.50–0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA‐based approach has the potential to become the next‐generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.     

Monitoring endangered freshwater biodiversity using environmental DNA

Freshwater ecosystems are among the most endangered habitats on Earth, with thousands of animal species known to be threatened or already extinct. Reliable monitoring of threatened organisms is crucial for data‐driven conservation actions but remains a challenge owing to nonstandardized methods that depend on practical and taxonomic expertise, which is rapidly declining. Here, we show that a diversity of rare and threatened freshwater animals—representing amphibians, fish, mammals, insects and crustaceans—can be detected and quantified based on DNA obtained directly from small water samples of lakes, ponds and streams. We successfully validate our findings in a controlled mesocosm experiment and show that DNA becomes undetectable within 2 weeks after removal of animals, indicating that DNA traces are near contemporary with presence of the species. We further demonstrate that entire faunas of amphibians and fish can be detected by high‐throughput sequencing of DNA extracted from pond water. Our findings underpin the ubiquitous nature of DNA traces in the environment and establish environmental DNA as a tool for monitoring rare and threatened species across a wide range of taxonomic groups.     

不同环境样本类型对蚌类环境DNA监测的差异研究

为了阐明在使用环境DNA宏条形码技术时不同环境样本类型如何影响蚌类物种的可检测性,于2021年冬季和春季在鄱阳湖分别采集表层水、底层水和沉积物进行环境DNA宏条形码分析,并结合传统方法采集验证。基于环境DNA宏条形码技术共检测到鄱阳湖蚌类33种,传统方法共采集蚌类18种,环境DNA宏条形码技术能检测出传统方法采集到的所有蚌类物种。表层水和底层水注释到的蚌类物种数均分别高于沉积物的,且表层水和底层水注释到的蚌类物种分别完全覆盖沉积物的。基于环境DNA宏条形码技术的蚌类α多样性水平季节差异不显著,但蚌类β多样性水平季节差异显著。表层水和底层水的蚌类多样性均显著高于沉积物样本的, Beta多样性分析也显示水体样本(表层水和底层水分别)和沉积物样本存在显著性差异。但表层水和底层水的蚌类多样性和群落结构均无显著差异。鄱阳湖蚌类群落结构与环境因子的关联分析表明水深(WD)、透明度(SD)、水温(WT)和总氮(TN)显著影响蚌类群落结构。环境DNA宏条形码技术在蚌类的多样性监测中可行,且采水样比采沉积物效果好,表层水和底层水无显著差异。     

Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofilters

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4–5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥81–86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types.     

Effect of Biotic and Abiotic Factors on In Vitro Proliferation, Encystment, and Excystment of Pfiesteria piscicida

Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4°C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.     

Estimation of fish biomass using environmental DNA

Environmental DNA (eDNA) from aquatic vertebrates has recently been used to estimate the presence of a species. We hypothesized that fish release DNA into the water at a rate commensurate with their biomass. Thus, the concentration of eDNA of a target species may be used to estimate the species biomass. We developed an eDNA method to estimate the biomass of common carp (Cyprinus carpio L.) using laboratory and field experiments. In the aquarium, the concentration of eDNA changed initially, but reached an equilibrium after 6 days. Temperature had no effect on eDNA concentrations in aquaria. The concentration of eDNA was positively correlated with carp biomass in both aquaria and experimental ponds. We used this method to estimate the biomass and distribution of carp in a natural freshwater lagoon. We demonstrated that the distribution of carp eDNA concentration was explained by water temperature. Our results suggest that biomass data estimated from eDNA concentration reflects the potential distribution of common carp in the natural environment. Measuring eDNA concentration offers a non-invasive, simple, and rapid method for estimating biomass. This method could inform management plans for the conservation of ecosystems.     

Detection of a diverse marine fish fauna using environmental DNA from seawater samples

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days.Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.     

Fish abundance and species richness across an estuarine–freshwater ecosystem in the Neotropics

We investigated the distributional patterns of shallow-water fish and their environmental correlates along a broad spatial scale encompassing estuarine and freshwater ecosystems. Marine-vagrant species were restricted to the sites under the influence of salinity intrusion, whereas estuarine-related and freshwater guilds were found along the entire estuarine–freshwater gradient. Primary- and secondary-freshwater fish guilds had the most widespread spatial distribution and comprised a major fraction of the total abundance and species richness. Abiotic factors correlated with fish abundance and composition along two main environmental axes, one related with variation in salinity, water transparency, and sediment granulometry and the other with the slope gradient. Species richness was significantly higher at the natural channel linking the estuarine- and freshwater-ecosystem, which probably was due to: (a) a steeper slope that favored the confluence of fish from the littoral (<2 m) and limnetic (>2 m) zones and (b) the sporadic inflow of saltwater that carried into this region several marine-related species. Although estuarine–freshwater ecotones are known to support few species, mainly salinity tolerant, our results suggest that habitat features and seasonal fish movement associated with salinity intrusion could lead to more diverse fish assemblages in this transitional zone.     

Marine water environmental DNA metabarcoding provides a comprehensive fish diversity assessment and reveals spatial patterns in a large oceanic area

Current methods for monitoring marine fish (including bony fishes and elasmobranchs) diversity mostly rely on trawling surveys, which are invasive, costly, and time‐consuming. Moreover, these methods are selective, targeting a subset of species at the time, and can be inaccessible to certain areas. Here, we used environmental DNA (eDNA), the DNA present in the water column as part of shed cells, tissues, or mucus, to provide comprehensive information about fish diversity in a large marine area. Further, eDNA results were compared to the fish diversity obtained in pelagic trawls. A total of 44 5 L‐water samples were collected onboard a wide‐scale oceanographic survey covering about 120,000 square kilometers in Northeast Atlantic Ocean. A short region of the 12S rRNA gene was amplified and sequenced through metabarcoding generating almost 3.5 million quality‐filtered reads. Trawl and eDNA samples resulted in the same most abundant species (European anchovy, European pilchard, Atlantic mackerel, and blue whiting), but eDNA metabarcoding resulted in more detected bony fish and elasmobranch species (116) than trawling (16). Although an overall correlation between fishes biomass and number of reads was observed, some species deviated from the common trend, which could be explained by inherent biases of each of the methods. Species distribution patterns inferred from eDNA metabarcoding data coincided with current ecological knowledge of the species, suggesting that eDNA has the potential to draw sound ecological conclusions that can contribute to fish surveillance programs. Our results support eDNA metabarcoding for broad‐scale marine fish diversity monitoring in the context of Directives such as the Common Fisheries Policy or the Marine Strategy Framework Directive.     

Genetic Analysis of Betanodaviruses in Subclinically Infected Aquarium Fish and Invertebrates

Betanodaviruses causing viral nervous necrosis (VNN) have been detected and isolated from several species of cultured marine fish worldwide. In Korea, VNN was identified in several species of cultured marine fish. This study presents data on the amplified nested PCR product (420 bp) of 11 nodavirus strains from different species of apparently healthy aquarium fish and invertebrates collected in one private commercial aquarium in Korea. Phylogenetic analyses based on the partial nucleotide sequence (177 bases) of the RNA2 coat protein gene were identical to the redspotted grouper nervous necrosis virus (RGNNV) genotype (96%–100%). The presence of the RGNNV type of betanodaviruses in these subclinically infected aquarium fish and invertebrates imported from different countries probably indicates that the samples were contaminated inside the aquarium and represents a serious challenge for its management of viral nervous necrosis. These positive samples can be an inoculum source of betanodavirus infection to other susceptible fish species inside the aquarium.