Ocean College, Zhejiang University, Zhoushan 316021, China
2.
Key Laboratory of Marine Ecosystem Dynamics, Second Institute of Oceanography, Ministry of Natural Resources, Hangzhou 310012, China
3.
The State Key Laboratory of Marine Pollution, Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR 999077, China
4.
Zhejiang Marine Ecology and Environment Monitoring Center, Zhoushan 316000, China
Funds:
The National Natural Science Foundation of China under contract Nos 41706191 and 41961144013; the Natural Science Foundation of Zhejiang Province under contract No. LY20D060004; the National Natural Science Foundation of China under contract Nos 41676111, 41876139 and 41906140; Program of Bureau of Science and Technology of Zhoushan grant under contract No. 2019C81031 and Basic Public Welfare Research Project of Zhejiang Province under contract No. LGC22B050032.
More than 30 species of benthic Prorocentrum have been identified, some of which produce okadaic acid (OA) and its derivatives, dinophysistoxins (DTXs), which cause diarrhetic shellfish poisoning (DSP). Increasing numbers of benthic Prorocentrum species have been reported in tropical and subtropical waters of the China Sea. In contrast, only a few benthic Prorocentrum species have been reported in temperate waters. In this study, morphological descriptions obtained using light microscopy, scanning electron microscopy and molecular characterization of one Prorocentrum clipeus strain isolated from the Yellow Sea of China are presented. Prorocentrum clipeus cells were nearly circular in shape, with a collar, ridge, and one protrusion. The periflagellar area was wide U-shaped, with two curved projections on platelet 1a. Nine periflagellar platelets of different sizes were observed. The morphology closely fits that of the species isolated from other locations. Phylogenetic analysis based on the molecular sequences of the small subunit (SSU) rDNA, internal transcribed spacer (ITS), and large subunit (LSU) rDNA was performed. A comprehensive metabolomic analysis incorporating target, suspect and non-target screenings was first applied to investigate the intracellular and extracellular metabolite profiles of the current isolate of P. clipeus. According to the results of the target and suspect screenings, 179 metabolites or toxins produced by DSP-related algal species, including OA, dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2) and pectenotoxin-2 (PTX2), were not detected. Non-target screening involving feature-based molecular networking (FBMN) provided a global view of major metabolites produced by the P. clipeus DF128 strain and revealed 23 clusters belonging to at least 13 compound classes, with organometallic compounds, lipids and lipid-like molecules, phenylpropanoids and polyketides, and benzenoids as major types. To date, this is the first record of the characterization of P. clipeus in samples from Chinese waters. Our results support the wide distribution of epibenthic Prorocentrum species.
Figure 1. Light microscopy (LM) and scanning electron microscopy (SEM) images of P.clipeus DF128. LM, right thecal view showing the cell shape, the large nucleus (N) posterior (a). Laser scanning confocal microscopy images of P. clipeus (b - d). Epifluorescence image showing the pyrenoid (Py) and radial arrangement of chloroplasts (Chl) (b). Sybr Green stained cell showing the shape of the nucleus (N) (c). Epifluorescence image of the thecal valve of the cell (d). SEM. The right valve view shows that the cell shape is asymmetrical and round (e). SEM. Left valve view showing the smooth thecal surface with a radial pore pattern (f). SEM. The intercalary band is wide and has transverse striation (g). SEM. The cell is shown in the right lateral view, with a wide arc-shaped periflagellar area. Ridge (asterisk), wing-shaped protrusion (arrow), curved projections (arrowhead), detail of the nine platelets (h and i). Scale bars in a-g: 10 μm, h and i: 5 μm.
Figure 2. Maximum likelihood tree of 33 SSU rDNA sequences and 1691 positions. Alexandrium tamarense was included as an outgroup. The best model, chosen by MrModel-Test2.3, was GTR+I+G. The support values shown were obtained by maximum likelihood and Bayesian inference. Only values > 50% (ML) and 0.50 (BI) are shown. A new sequence published in this study is displayed in bold (OP601437).
Figure 3. Maximum likelihood tree of 32 ITS1–5.8S–ITS2 sequences and 623 positions. Karenia brevis was included as an outgroup. The best model, chosen by MrModel-Test2.3, was GTR+I+G. The support values shown were obtained by maximum likelihood and Bayesian inference. Only values > 50% (ML) and 0.50 (BI) are shown. A new sequence published in this study is displayed in bold (OP601439).
Figure 4. Maximum likelihood tree of 37 LSU rDNA sequences and 697 positions. Alexandrium tamarense was included as an outgroup. The best model, chosen by MrModel-Test2.3, was GTR+I+G. The support values shown were obtained by maximum likelihood and Bayesian inference. Only values > 50% (ML) and 0.50 (BI) are shown. A new sequence published in this study is displayed in bold (OP601441).
Figure 5. Extracted ion chromatograms (EICs) of the procedure blank; P. clipeus DF128 intracellular and extracellular extracts; and OA, DTX1 and DTX2 standards (100 ng/mL) in negative ESI mode using a Sciex QTRAP 5 500 system.
Figure 6. The EICs of the procedural blank, P. clipeus DF128 intracellular and extracellular extracts, and standard PTX2 (100 ng/mL) in positive ESI mode were determined using a Sciex QTRAP 5 500 system.
Figure 7. Enhanced molecular networks obtained from the positive ion mode (A) and negative ion mode (B) mass spectra using MolNetEnhancer showing different molecular families/clusters of the pooled metabolites in the extracts of P. clipeus DF128. The node colors represent the classes of putatively annotated metabolites matched in the GNPS libraries. Single nodes indicate the absence of MS/MS fragments shared with any other compound.
Figure 8. Global occurrence of P. clipeus (data from this paper indicated by double circle marker; data from published literature and OBIS from 2000 to 2019 in black). The figure was created using Ocean Data View, version 5.7.1(Schlitzer, 2023).
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