SPECIES DETECTION: CNIDARIANS

Shinzato C., Zayasu Y., Kanda M., Kawamitsu M., Satoh N., Yamashita H., Suzuki G. (2018): Using seawater to document coral-zoothanthella diversity: a new approach to coral reef monitoring using environmental DNA. Frontiers in Marine Science 5: 28.
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Frequent, high-density coral monitoring is essential to understand coral reef ecosystems. For this purpose, we developed a novel method for simultaneous monitoring of Acropora corals and their symbiont, Symbiodinium, from environmental DNA (eDNA) in seawater using next generation sequencing technology (NGS). We performed a tank experiment with running seawater using 19 Acropora species. Complete mitochondrial genomes of all the Acropora species were assembled to create a database and major types of their Symbiodinium symbionts were identified. Then eDNA was isolated by filtering inlet and outlet seawater from the tanks. Acropora and Symbiodinium DNA were amplified by PCR and sequenced. We detected all of the tested Acropora types from eDNA samples. Proportions and numbers of DNA sequences were both positively correlated with masses of corals in the tanks. In this trial, we detected DNA sequences from as little as 0.04 kg of Acropora colony, suggesting that existence of at least one adult Acropora colony (~30 cm diameter = 1 kg) per m2 at depths < 10 m could be detected using eDNA in the field. In addition, we detected major types of Symbiodinium within host corals from seawater, suggesting that it should be possible to detect major coral symbiont types if Acropora corals exist nearby, and possible free-living state Symbiodinium cells from eDNA in seawater. eDNA abundance of Symbiodinium types did not correlate well with frequencies of major Symbiodinium types in the corals, suggesting that quantification of Symbiodinium is difficult at this stage. Although this is the initial attempt to detect coral and Symbiodinium simultaneously from eDNA in seawater, this method may allow us to perform high-frequency, high-density coral reef monitoring of both corals and their symbionts in the near future.

Nichols P. K., Marko P. B. (2019): Rapid assessment of coral cover from environmental DNA in Hawai’i. Environmental DNA 1: 40-53.
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Coral reefs support the most diverse assemblages of marine life on Earth, yet are declining due to local and global stressors. Rapid and widespread monitoring is essential for tracking ecosystem responses, but assessment of coral communities traditionally relies on time‐consuming visual estimates of coral cover, the percentage of substrate occupied by living corals. The analysis of environmental DNA (eDNA) offers fast and efficient insights into the abundance and distribution of species, yet it remains untested to monitor coral biomass. Here, we demonstrate that visual estimates are highly correlated with the abundance of coral eDNA on reefs in Hawai’i measured with a relatively simple, rapid, but replicated PCR‐based metabarcoding approach. Target sequence length was also tested by amplifying short (~120 base‐pairs) and long (~400 base‐pairs) fragments from the same region of two mitochondrial DNA genes, 16S ribosomal DNA, and cytochrome oxidase‐1 using primers designed to preferentially amplify Hawaiian coral genera. Careful primer selection and target sequence lengths play an important role in determination of coral abundance from eDNA biomass. Given its broad applicability and ease of use, eDNA metabarcoding can provide complementary analytical support for biomonitoring programs and management initiatives tracking changes in coral cover caused by climate change and other disturbances on coral reefs.

Shinzato C., Narisoko H., Nishitsuji K., Nagata T., Satoh N., Inoue J. (2021): Novel mitochondrial DNA markers for scleractinian corals and generic-level environmental DNA metabarcoding. Frontiers in Marine Science 8: 758207.
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Coral reefs, the most biodiverse habitats in the ocean, are formed by anthozoan cnidarians, the scleractinian corals. Recently, however, ongoing climate change has imperiled scleractinian corals and coral reef environments are changing drastically. Thus, convenient, high-density monitoring of scleractinian corals is essential to understand changes in coral reef communities. Environmental DNA (eDNA) metabarcoding is potentially one of the most effective means of achieving it. Using publicly available scleractinian mitochondrial genomes, we developed high-specificity primers to amplify mitochondrial 12S ribosomal RNA (12S) and cytochrome oxidase-1 (CO1) genes of diverse scleractinian corals, which could be used for genus-level metabarcoding analyses, using next-generation sequencing technologies. To confirm the effectiveness of these primers, PCR amplicon sequencing was performed using eDNA isolated along the seashore of Okinawa, Japan. We successfully amplified all eDNA samples using PCR. Approximately 93 and 72% of PCR amplicon sequences of 12S and CO1 primers originated from scleractinian 12S and CO1 genes, respectively, confirming higher specificities for coral mitochondrial genes than primers previously used for coral eDNA metabarcoding. We also found that hierarchical clustering, based on the percentage of mapped reads to each scleractinian genus, discriminates between sampling locations, suggesting that eDNA surveys are sufficiently powerful to reveal differences between coral communities separated by <1 km. We conclude that the method reported here is a powerful tool for conducting efficient eDNA surveys targeting scleractinian corals.

Dugal L., Thomas L., Wilkinson S. P., Richards Z. T., Alexander J. B., Adam A. A., Kennington W. J., Jarman S., Ryan N. M., Bunce M., Gilmour J. P. (2022): Coral monitoring in northwest Australia with environmental DNA metabarcoding using a curated reference database for optimized detection. Environmental DNA 4: 63-76.
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The need for efficient and more accurate ways of monitoring threatened ecosystems is becoming increasingly urgent as climate change intensifies. Coral reefs are an example of an ecosystem in crisis, with widespread declines in coral cover and diversity documented over recent decades. Novel molecular approaches such as biomonitoring using environmental DNA (eDNA) from seawater samples show great potential to complement future coral reef monitoring programs, especially when used in combination with conventional methods. However, eDNA metabarcoding studies often rely on public databases (e.g., GenBank) for assigning taxonomy, which generally limits the number of sequences that can be taxonomically identified. The extent to which building reference tissue sequences improves taxonomic resolution has yet to be fully examined. Here, we combined traditional coral reef monitoring data with eDNA assessments derived from seawater collected at the highly diverse Rowley Shoals in Western Australia. Using two ITS2 assays developed to target basal metazoan DNA and a reference database spiked with 70 local coral specimens, we identified 37 genera and 40 species from 56 1 L seawater samples. We identified considerable overlap of taxa with visual survey data and showed that assignment of amplicon sequence variants was significantly improved when “spiking” the taxonomic classifier with curated sequences of locally collected species. Our findings showcase the potential of eDNA metabarcoding for monitoring the biodiversity of reef corals and highlight the importance of custom reference sequence databases for improving taxonomic resolution in metabarcoding studies.

Ip Y. C. A., Chang J. J. M., Tun K. P. P., Meier R., Huang D. (2022): Multispecies environmental DNA metabarcoding sheds light on annual coral spawning events. Molecular Ecology 32: 6474-6488.
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Synchronous multispecific coral spawning generally occurs annually and forms an integral part of the coral life cycle. Apart from spawning times and species participation, however, much else remains unknown. Here, we applied environmental DNA (eDNA) metabarcoding to study two tropical reef sites of contrasting coral cover before, during and after coral spawning. Using coral-ITS2 and vertebrate-12S markers, we evaluated eDNA as an alternative monitoring tool by assessing its capabilities in detecting spawning species and tracking relative abundances of coral and fish eDNA. Over 3 years, elevated eDNA coral signals during the event (proportional read increase of up to five-fold) were observed, detecting a total of 38 coral and 133 fish species with all but one of the coral species visually observed to be spawning. This is also the first demonstration that eDNA metabarcoding can be used to infer the diurnal partitioning of night- and day-time spawning, spawning in coral species overlooked by visual surveys, and the associated changes in fish trophic structures as an indicator of spawning events. Our study paves the way for applied quantitative eDNA metabarcoding approaches to better study ephemeral and important biological events.

Gösser F., Schweinsberg M., Mittelbach P., Schoenig E., Tollrian R. (2023): An environmental DNA metabarcoding approach versus a visual survey for reefs of Koh Pha‐ngan in Thailand. Environmental DNA 5: 297-311.
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Information on diversity indices and abundance of individual species is crucial for the assessment of ecosystem health, especially for endangered ecosystems as coral reefs. The application of environmental DNA (eDNA) to monitor coral biodiversity is, however, just beginning to come into focus for marine biologists. In this study, an eDNA metabarcoding approach of seawater samples in three different reefs on Koh Pha-ngan, Thailand, was compared with simultaneously collected visual census data. In addition, differences in read abundance and number of genera detected between daytime and nighttime eDNA samples were examined, and a local coral barcode reference database (n = 23 genera; COI gene) was constructed to improve assignment of eDNA reads to the genus level. As a technical extension of existing assays, two methods for library construction were compared: a commercial kit and in-house developed fusion primers. Combining eDNA metabarcoding and visual data, 29 different genera of scleractinian corals from 14 families were detected. In addition, a log-linear correlation was found between the abundance of eDNA reads and visually determined relative coral cover at the genus level, suggesting a predictive relationship between eDNA reads and coral cover. Results also showed diurnal variation between day and night samples in the number of eDNA reads, purported to relate to the activity phases of corals. The use of uniquely labeled fusion primers, gave comparable results to a commercially available library preparation kit. Especially with frequent use, fusion primers can be very cost-effective, and therefore a consideration for large-scale studies. Using a custom reference database of 89 sequences from coral tissue samples of 23 different coral genera produced better results than querying against NCBI GenBank, highlighting the importance of locally optimized databases. We consider these results important for establishing eDNA as a complementary tool to visual surveys to track changes in coral diversity and cover.

JELLYFISH

Minamoto T., Fukuda M., Katsuhara K. R., Fujiwara A., Hidaka S., Yamamoto S., Takahashi K., Masuda R. (2017): Environmental DNA reflects spatial and temporal jellyfish distribution. Plos One 12: e0173073.
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Recent development of environmental DNA (eDNA) analysis allows us to survey underwater macro-organisms easily and cost effectively; however, there have been no reports on eDNA detection or quantification for jellyfish. Here we present the first report on an eDNA analysis of marine jellyfish using Japanese sea nettle (Chrysaora pacifica) as a model species by combining a tank experiment with spatial and temporal distribution surveys. We performed a tank experiment monitoring eDNA concentrations over a range of time intervals after the introduction of jellyfish, and quantified the eDNA concentrations by quantitative real-time PCR. The eDNA concentrations peaked twice, at 1 and 8 h after the beginning of the experiment, and became stable within 48 h. The estimated release rates of the eDNA in jellyfish were higher than the rates previously reported in fishes. A spatial survey was conducted in June 2014 in Maizuru Bay, Kyoto, in which eDNA was collected from surface water and sea floor water samples at 47 sites while jellyfish near surface water were counted on board by eye. The distribution of eDNA in the bay corresponded with the distribution of jellyfish inferred by visual observation, and the eDNA concentration in the bay was ~13 times higher on the sea floor than on the surface. The temporal survey was conducted from March to November 2014, in which jellyfish were counted by eye every morning while eDNA was collected from surface and sea floor water at three sampling points along a pier once a month. The temporal fluctuation pattern of the eDNA concentrations and the numbers of observed individuals were well correlated. We conclude that an eDNA approach is applicable for jellyfish species in the ocean.

Martin-Abadal M., Ruiz-Frau A., Hinz H., Gonzalez-Cid Y. (2020): Jellytoring: real-time jellyfish monitoring based on deep learning object detection. Sensors 20: 1708.
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During the past decades, the composition and distribution of marine species have changed due to multiple anthropogenic pressures. Monitoring these changes in a cost-effective manner is of high relevance to assess the environmental status and evaluate the effectiveness of management measures. In particular, recent studies point to a rise of jellyfish populations on a global scale, negatively affecting diverse marine sectors like commercial fishing or the tourism industry. Past monitoring efforts using underwater video observations tended to be time-consuming and costly due to human-based data processing. In this paper, we present Jellytoring, a system to automatically detect and quantify different species of jellyfish based on a deep object detection neural network, allowing us to automatically record jellyfish presence during long periods of time. Jellytoring demonstrates outstanding performance on the jellyfish detection task, reaching an F1 score of 95.2%; and also on the jellyfish quantification task, as it correctly quantifies the number and class of jellyfish on a real-time processed video sequence up to a 93.8% of its duration. The results of this study are encouraging and provide the means towards a efficient way to monitor jellyfish, which can be used for the development of a jellyfish early-warning system, providing highly valuable information for marine biologists and contributing to the reduction of jellyfish impacts on humans.

Ames C. L., Ohdera A. H., Colston S. M., Collins A. G., Fitt W. K., Morandini A. C., Erickson J. S., Vora G. J. (2021): Fieldable environmental DNA sequencing to assess jellyfish biodiversity in nearshore waters of the Florida Keys, United States. Frontiers in Marine Science 8: 369. 
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Recent advances in molecular sequencing technology and the increased availability of fieldable laboratory equipment have provided researchers with the opportunity to conduct real-time or near real-time gene-based biodiversity assessments of aquatic ecosystems. In this study, we developed a workflow and portable kit for fieldable environmental DNA sequencing (FeDS) and tested its efficacy by characterizing the breadth of jellyfish (Medusozoa) taxa in the coastal waters of the Upper and Lower Florida Keys. Environmental DNA was isolated from seawater collection events at eight sites and samples were subjected to medusozoan 16S rRNA gene and metazoan mitochondrial cytochrome oxidase 1 gene profiling via metabarcoding onsite. In total, FeDS yielded 175,326 processed sequence reads providing evidence for 53 medusozoan taxa. Our most salient findings revealed eDNA from: (1) two venomous box jellyfish (Cubozoa) species, including taxa whose stings cause the notorious Irukandji envenomation syndrome; (2) two species of potentially introduced stalked jellyfish (Staurozoa); and (3) a likely cryptic species of upside-down jellyfish (Scyphozoa). Taken together, the results of this study highlight the merits of FeDS in conducting biodiversity surveys of endemic and introduced species, and as a potential tool for assessing envenomation and/or conservation-related threats.

Bolte B., Goldsbury J., Huerlimann R., Jerry D., Kingsford M. (2021): Validation of eDNA as a viable method of detection for dangerous cubozoan jellyfish. Environmental DNA 3: 769-779.
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Stings from certain species of cubozoan jellyfish are dangerous to humans and their seasonal presence in tropical marine waters poses a significant risk to coastal communities. The detection of cubozoans is difficult due to high spatial and temporal variation in their occurrence and abundance. Environmental DNA (eDNA) has the potential to detect rare species and therefore offers potential to detect cubozoans, not only pelagic medusae, but presence of cryptic polyp life stages. The objective of this study was to validate the use of eDNA as a viable detection method for four cubozoan species (Chironex fleckeri, Copula sivickisi, Carybdea xaymacana, and Carukia barnesi). Species-specific primers were developed for each of these four cubozoans and an eDNA approach validated utilizing both laboratory and field trials. Laboratory DNA degradation experiments demonstrated that C. sivickisi DNA degraded quickly but could still be detected in sea water for up to 9 days post-jellyfish removal. Positive detection was found for C. fleckeri, C. xaymacana, and C. sivickisi medusae in the waters surrounding Magnetic Island, Queensland, in the Austral spring/summer (September-January). Based on visual surveys, there was a poor relationship between concentration of eDNA and abundance of jellyfish. Positive eDNA amplification was also found from water sampled near the substratum when C. sivickisi medusae were out of season and absent. This suggests the eDNA analysis was likely detecting C. sivickisi polyps located within the substratum. Consequently, eDNA is an effective tool to detect both the medusae and likely polyps of cubozoans. This approach provides the means to reduce the risk of envenomation to swimmers and enhance our knowledge of cubozoan ecology.

Choi S. Y., Kim H. J., Seo M. H., Soh H. Y. (2021): Density estimation of Nemopilema nomurai (Scyphozoa, Rhizostomeae) using a drone. Journal of the Indian Society of Remote Sensing 49: 1727-1732.
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Research to understand the distribution and density of jellyfish is actively being conducted using training ships, but this is hindered by the high cost of manpower and the limitations of the irradiation area. Unmanned aerial vehicles (UAVs or drones), however, provide cost-effective means for assessing marine animal populations. Therefore, we tested the application of UAVs in estimating jellyfish density and probed the altitude-dependent suitability of these devices. We analyzed images obtained by a drone as well as by manual counting and used ImageJ to measure the density of Nemopilema nomurai off Sang-Chuja Island, Jeju, South Korea. Analysis of the image obtained at altitudes of 5–120 m allowed for the identification of 2–173 individuals, while 1.49–9.09 individuals were identified per 100 m2. Jellyfish density data measured by manual count and by ImageJ did not show any difference below 90 m; however, a difference was presented at altitudes of 100 m (98%) and 120 m (95%). These results demonstrate the potential of drones for jellyfish monitoring and recommend an optimal altitude for observation.

Mcilwaine B., Casado M. R. (2021): JellyNet: The convolutional neural network jellyfish bloom detector. International Journal of Applied Earth Observation and Geoinformation 97: 102279.
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Coastal industries face disruption on a global scale due to the threat of large blooms of jellyfish. They can decimate coastal fisheries and clog the water intake systems of desalination and nuclear power plants. This can lead to losses of revenue and power output. This paper presents JellyNet: a convolutional neural network (CNN) jellyfish bloom detection model trained on high resolution remote sensing imagery collected by unmanned aerial vehicles (UAVs). JellyNet provides the detection capability for an early (6–8 h) bloom warning system. 1539 images were collected from flights at 2 locations: Croabh Haven, UK and Pruth Bay, Canada. The training/test dataset was manually labelled, and split into two classes: ‘Bloom present’ and ‘No bloom present’. 500 × 500 pixel images were used to increase fine-grained pattern detection of the jellyfish blooms. Model testing was completed using a 75/25% training/test split with hyperparameters selected prior to model training using a held-out validation dataset. Transfer learning using VGG-16 architecture, and a jellyfish bloom specific binary classifier surpassed an accuracy of 90%. Test model performance peaked at 97.5% accuracy. This paper exhibits the first example of a high resolution, multi-sensor jellyfish bloom detection capability, with integrated robustness from two oceans to tackle real world detection challenges.

Morrissey S. J., Jerry D. R., Kingsford M. J. (2022): Genetic detection and a method to study the ecology of deadly cubozoan jellyfish. Diversity 14: 1139.
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Cubozoan jellyfish pose a risk of envenomation to humans and a threat to many businesses, yet crucial gaps exist in determining threats to stakeholders and understanding their ecology. Environmental DNA (eDNA) provides a cost-effective method for detection that is less labour intensive and provides a higher probability of detection. The objective of this study was to develop, optimise and trial the use of eDNA to detect the Australian box jellyfish, Chironex fleckeri. This species was the focus of this study as it is known to have the strongest venom of any cubozoan; it is responsible for more than 200 recorded deaths in the Indo-Pacific region. Further, its ecology is poorly known. Herein, a specific and sensitive probe-based assay, multiplexed with an endogenous control assay, was developed, and successfully utilised to detect the deadly jellyfish species and differentiate them from closely related taxa. A rapid eDNA decay rate of greater than 99% within 27 h was found with no detectable influence from temperature. The robustness of the technique indicates that it will be of high utility for detection and to address knowledge gaps in the ecology of C. fleckeri; further, it has broad applicability to other types of zooplankton.