Biodiversity Data Journal :
Taxonomy & Inventories
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Corresponding author: Jinsoon Park (jpark@kmou.ac.kr)
Academic editor: Dimitris Poursanidis
Received: 06 Jan 2023 | Accepted: 28 May 2023 | Published: 17 Jun 2023
© 2023 Sungha Cho, Inho Yang, Jong Seong Khim, Jinsoon Park
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Cho S, Yang I, Khim JS, Park J (2023) First confirmed report of Nassarius sinarum (Mollusca, Gastropoda) in Korea. Biodiversity Data Journal 11: e99661. https://doi.org/10.3897/BDJ.11.e99661
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The marine gastropod mollusc Nassarius sinarum has attracted attention due to its status as a potential invasive species and the ecological impact it may have on local environments and the fishing industry. It was observed exclusively within China initially, but its distribution now seems to have expanded into Japan and Korea. Accurate identification of N. sinarum, particularly in its juvenile stage, is vital for understanding its ecological influences and distribution patterns.
This study represents the first comprehensive analysis of N. sinarum samples from Korea. It includes morphological examination, scanning electron microscopy images and molecular sequencing. Two live specimens were collected from the Yeongsan River estuary in Korea and their morphological features were analysed and compared to those of samples from China and Japan. The samples’ species were confirmed by molecular identification, based on cytochrome c oxidase subunit I (COI) and histone H3 (H3) genetic markers.
It was observed that juvenile N. sinarum shells lack key species-characteristic morphological traits, such as a thick outer lip and diminishing axial ribs. However, COI marker-based molecular identification affirmed that these Korean specimens were N. sinarum. The H3 region was registered with the National Center for Biotechnology Information (NCBI) for the first time. Phylogenetic analysis of the H3 region did not resolve species distinctions within the Nassarius, suggesting that the H3 marker is not suitable for species identification within this genus. In this context, multiple genetic markers, when used appropriately, can also be applied to genus-level searches, enhancing species identification accuracy and reducing misidentification.
The sequences provided in this study can serve as a valuable reference for future DNA barcoding research. Additional samples and surveys should be conducted through collaborative efforts amongst national and institutional organisations to further clarify the ecological status of N. sinarum and to investigate its distribution and potential impact around East Asia. Finally, a new Korean name, (No-lan-jul-job-ssal-mu-nui-go-dung; 노란줄좁쌀무늬고둥) has been proposed for N. sinarum.
COI, DNA barcoding, estuary, Gastropoda, H3, scanning electron microscope
The Nassariidae constitute a globally-occurring family of shelled gastropods with a broad habitat range spanning from the intertidal zone to deep waters (1000 m depth) and with a distribution that encompasses temperate, subtropical, tropical and cold waters (
For more than a century and a half after its initial discovery, the Nassariidae species Nassarius sinarum (Philippi 1851) was observed solely in the China Sea (
The sole documentation of N. sinarum in Korea prior to the present study was a solitary photograph without morphological verification of the specimen, DNA barcoding, a record of whether the specimen was a whole organism or merely a shell or the vouchering of the specimen to a museum. Furthermore, there were morphological discrepancies compared to previously-reported N. sinarum individuals, particularly with respect to the aperture. Thus, the purported initial identification of N. sinarum in Korea requires more accurate evidence to be collected. These inadequacies may account for the species' absence in Korean records.
In the present study, specimens of N. sinarum were collected from the Yeongsan River estuary in Korea. A detailed assessment of N. sinarum was conducted, including morphological description and verification, detailed structural and scanning electron microscopy and molecular verification. Nassarius species are usually distinguished, based on shell morphology, especially shell sculpture. However, this distinction might be imprecise and lead to confusion (
In June 2022, two Nassarius sinarum specimens were obtained from the Yeongsan River estuary, situated in the southwest coastal region of the Korean Peninsula. Following collection, the specimens were preserved immediately at -80°C in a 50 ml conical tube containing seawater from the site to minimise any alterations to their genetic material. This preservation method was adhered to scrupulously to prevent any adverse pre-extraction effects on DNA.
The first specimen (labelled as "A") was subjected to analysis of muscle tissue, which was obtained after breaking its shell for DNA examination. Due to the specimen's diminutive size, extensive shell damage was necessary to isolate muscle tissue for genetic analysis. Subsequently, a sequence file for sample A was submitted to the NCBI, a repository for DNA barcode records related to GenBank data. The submission, which included an extensive data file, underwent a meticulous review process employing BankIt tools to ensure proper formatting, sequence quality and the absence of potential contamination. The data file contained details regarding the organism, organelles, isolation source, country of origin, coordinates, collection date, collectors and identifiers. The submission also included a trace file that indicated the primers used, sequence directionality and molecular marker information. COI and H3 sequences were edited, aligned in FASTA format, assigned a Sample ID and uploaded to GenBank. The specimens' shells were inspected under a Leica S9D stereomicroscope, equipped with a K3C camera.
To prepare it for scanning electron microscopy observation, the second specimen (labelled as "B") was treated with a 10% sodium hydroxide (NaOH) solution at 40°C for 48 hours and then rinsed thoroughly with distilled water. The shells then underwent five consecutive ultrasonic cleaning cycles, each lasting 10 minutes, before being dried completely in a desiccator at 40°C and mounted on stubs for gold coating and scanning electron microscopy analysis. The examination was conducted using a MIRA-3 Field Emission Scanning Electron Microscope (Tescan, Brno, Czech Republic) with aluminium stubs secured with carbon tape. Upon completion of scanning electron microscopy imaging, the pristine N. sinarum shell of Sample B was deposited at the Korea Marine Biodiversity Research Institute (MABIK, MO00184324). DNA analysis could not be performed on Sample B due to the requirement of shell crushing for DNA extraction and the dissolution of all organic matter for scanning electron microscopy imaging.
We used a HiGene Genomic DNA prep kit (Biofact, Daejeon, Korea) to isolate genomic DNA from 2 mg muscle tissue samples. Extracted DNA was subjected to bidirectional sequencing, focusing on two DNA regions for amplification: COI and H3. COI was amplified with forward and reverse primers:
LCO1490 (5’-GTAAAACGACGGCCAGTGGTCAACAAATCATAAAGATATTGG-3’) and
HCO2198 (5'-CAGGAAACAGCTATGACTAAACTTCAGGGTGACCAAAAAATC A-3')
(
H3aF (5'-ATGGCTCGTACCAAGCAGAC(ACG)GC-3') and
H3aR (5'-ATATCCTT(AG)GGCAT(AG)AT(AG)GTG AC-3') (
For COI gene amplification, a 25 μl reaction mixture was prepared consisting of 2 μl DNA template, 12.5 μl 2X Lamp Taq PCR Master mix (Biofact) and 2 μl of forward and reverse primers mixed together (each 10 pmol/μl). Similarly, H3 was amplified with a 25 μl reaction mixture containing 2 μl genomic DNA template, 2.5 μl 10X Lamp Taq buffer, 0.5 μl 10 mM dNTP mix, 0.4 μl Lamp Taq PCR Master mix (Biofact) and 2 μl of forward and reverse primers mixed together (each 10 pmol/μl). The final volume of each of these two reaction mixtures was adjusted to 25 μl with distilled water.
The thermocycling protocol involved separate PCR procedures for COI and H3. The COI PCR was comprised of an initial 120-s denaturation step at 95°C, followed by forty 20-s denaturation cycles, a 40-s annealing step at 45°C, a 60-s extension step at 72°C and a final 300-s extension step at 72°C. The H3 PCR consisted of an initial 120-s denaturation step at 95°C, followed by forty 20-s denaturation cycles, a 40-s annealing at 48°C, a 60-s extension step at 72°C and a final 180-s extension step at 72°C. Amplified PCR products were visualised using agarose gel electrophoresis (1% agarose gel) under UV light after staining with EcoDye™ DNA Staining Solution (Biofact). Sequencing reactions were carried out with BigDye™ Terminator V.3.1 Cycle Sequencing Kits (ABI, Waltham, USA) and all PCR products were sequenced using a 3730XL DNA Analyzer (ABI) at the Biofact sequencing facility.
In the construction of the COI phylogeny, we employed the 10 sequences of N. sinarum available on the NCBI database, along with 29 sequences from Nassarius species, culminating in a collective set of 43 sequences, inclusive of the three outgroup species. When it came to the H3 sequence of N. sinarum, we were limited by the fact that no other sequences from the same species were available, given our pioneering registration of it with the NCBI. Therefore, our analysis solely incorporated the sequences from the 29 chosen Nassarius species, bringing the total to 33 sequences when considering the three outgroup species. We have chosen these 29 Nassarius sequences from species that have sequences available for both COI and H3, thereby ensuring a consistent comparison under uniform conditions. Three species served as outgroups: Volutharpa perryi, Buccinum pemphigus and Neptunea cumingi (
The data underpinning the analysis reported in this paper are deposited in the Mendeley Data Repository at http://dx.doi.org/10.17632/zrb238r89g.2.
Buccinum sinarum (
Nassa (Niotha) sinarum (
Nassarius (Zeuxis) sinarus (
Nassarius (Tritonella) semiplicatus (
Nassarius (Zeuxis) semiplicatus (
Nassarius semiplicatus (
Zeuxis semiplicata (
On 16 June 2022, two live specimens were collected from the Yeongsan River estuary at Jeollanam-do, Korea (34°46'48"N, 126°26'31"E), located at 219 Daeburyeok-ro Samho-eup, Yeongam-gun (Fig.
Sample collection site details. A The fine-scale location of the collection site was obtained using Ocean Data View (Schlitzer R, 2015, http://odv.awi.de); B Photograph of the sampling location at the Yeongsan River, taken in June 2022 and provided by Sungha Cho.
Sample A, exhibiting a shell length of 14 mm and Sample B, with a shell length of 15 mm, are not fully developed individuals in comparison to the known maximum size of N. sinarum, which reaches up to 20 mm (Fig.
Morphological images of Nassarius sinarum from Korea, China and Japan. A & B Specimens collected alive in the estuary at the mouth of the Yeongsan River in Korea (this study) and considered to be juvenile shells; apertural and dorsal views were photographed; C Specimen collected alive in China; D Specimen collected alive in the mudflats of Japan; morphological features of the apertural and dorsal views suggest it is an adult shell. Scale bars for A and B are 5 mm; scale bars for C and D are not provided. For original images and data, consult Suppl. material
Scanning electron microscope images of the shell of Nassarius sinarum Specimen B, collected in this study. A Morphology, characterised by the absence of a developed callus and tooth in the aperture; B Axial ribs exhibiting a consistent appearance as they extend to the outer lip; C Body whorl sculpture; D Protoconch. Scale bar A & B = 2 mm, C = 1 mm, D = 200 μm. For additional scanning electron microscopy images, please refer to Suppl. material
Initially, N. sinarum was thought to be exclusive to the Yangtze River in China (
The sequence files were submitted to the NCBI and assigned accession numbers: OP693482 (660 bp) for COI and OP719775 (372 bp) for H3. The NCBI Basic Local Alignment Search Tool (BLAST) analysis demonstrated that our N. sinarum COI sequence matches that previously registered for N. sinarum, exhibiting 98.48–99.69% sequence similarity (Table
The top 10 results for each of the NCBI BLAST searches, conducted on the COI and H3 regions of Nassarius sinarum, are presented in descending order of percentage similarity.
COI |
H3 |
||||
BLAST search |
Accession No. (bp) |
Percent Similarity |
BLAST search |
Accession No. (bp) |
Percentage Similarity |
Nassarius sinarum |
KY100629 (642) |
99.69% |
Nassarius siquijorensis |
HQ834169 (354) |
100.00% |
Nassarius sinarum |
MG679291 (601) |
99.67% |
Nassarius cf. comptus |
KY489320 (331) | 100.00% |
Nassarius sinarum |
KY100644 (642) |
99.53% |
Nassarius velatus |
LC384075 (291) | 100.00% |
Nassarius sinarum |
KY100628 (642) |
99.53% |
Nassarius velatus |
LC384073 (291) | 100.00% |
Nassarius sinarum |
MG679294 (601) |
99.50% |
Nassarius velatus |
LC384072 (291) | 100.00% |
Nassarius sinarum |
MG679292 (601) |
99.50% |
Nassarius velatus |
LC384074 (283) | 100.00% |
Nassarius sinarum |
MG679290 (601) |
99.50% |
Nassarius conoidalis |
HQ834165 (354) |
99.72% |
Nassarius sinarum |
KY783918 (709) |
99.39% |
Nassarius sp. 279 |
KY489360 (331) |
99.70% |
Nassarius sinarum |
KY100642 (642) |
99.38% |
Nassarius arcularia |
KY489345 (331) | 99.70% |
Nassarius sinarum |
KY100638 (642) |
99.38% |
Nassarius moolenbeeki |
KY489339 (331) | 99.70% |
Maximum Likelihood tree of COI sequences of Nassarius sinarum collected in Korea. Bars represent genetic distances, letters preceding scientific names are NCBI accession numbers. A phylogenetic tree analysis conducted with the COI region demonstrates that N. sinarum has a distinctive species-indicative COI sequence.
Maximum Likelihood tree of H3 sequences of Nassarius sinarum collected in Korea. Bars represent genetic distances, letters preceding scientific names are NCBI accession numbers. A phylogenetic tree analysis conducted with the H3 region reveals that H3 sequences of N. sinarum cannot be used to clearly distinguish it from other Nassarius species.
Nassarius, commonly known as nassa mud snails (USA) or dog whelks (UK), is a genus of small to medium-sized marine gastropod molluscs in the Nassariidae characterised by their scavenging behaviour. These shelled gastropods inhabit mud flats and sand flats, have a global distribution and have a diverse habitat range that spans from the intertidal zone to deep waters (depths of at least 1000 m) in temperate, subtropical, tropical and cold-water environments. Nassarius species are highly active scavengers, feeding on crabs and carrion, such as dead fish. They often burrow into marine substrates, waiting with only their siphon exposed until detecting nearby food sources. Consistent with these ecological traits, N. sinarum inhabits both sandy and muddy mudflats and occupies various environments within the intertidal zone (
This study is the first to present a morphological analysis, inclusive of scanning electron microscopy images and molecular sequencing, of Nassarius sinarum individuals collected in Korea. The main identification features of N. sinarum include a thick outer lip and axial ribs that diminish towards the outer lip. Typically, juvenile Nassarius shells lack a fully developed callus, which leads to confusion in species identification (
The COI region of DNA, a universal barcode for gastropods, can be used to identify N. sinarum specimens. We registered the H3 region of the N. sinarum genome with NCBI for the first time and conducted a genus-level phylogenetic analysis. The phylogeny of the COI region formed a clear clade of COI-sequence conserved species with no regional variation amongst specimens in Korea, China and Japan (Fig.
This study was jointly supported by multiple grants. Firstly, the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1I1A2066477 for J. Park; NRF-2022R1C1C1008465 for I. Yang). Additionally, the research was backed by the Korea Institute of Marine Science & Technology (KIMST) with funding from the Ministry of Oceans and Fisheries. Two projects from KIMST were instrumental: "Development of risk managing technology tackling ocean and fisheries crisis around Korean Peninsula by Kuroshio Current" (RS-2023-00256330) and "Monitoring environmental and ecological changes in dynamic coasts and estuaries of the Korean four major rivers" (RS-2023-00238486). This paper was edited by a professional scientific editor at Write Science Right.
Original high-resolution images of live shells, dead shells and YSI; SEM images depicting sculpture details, protoconch, spire cross-section, columellar folds and posterior canal of Nassarius sinarum shells.