Morphology, molecular phylogeny and okadaic acid production of epibenthic Prorocentrum (Dinophyceae) species from the northern South China Sea

Around 30 epibenthic Prorocentrum species have been described, but information about their biogeography is limited. Some species are able to produce okadaic acid (OA) and its derivatives, which are responsible for diarrheic shellfish poisoning (DSP). In the present study, we examined the diversity of epibenthic Prorocentrum in the northern South China Sea by isolating single cells from sand, coral, and macroalgal samples collected from 2012 to 2015. Their morphology was examined using light microscopy and scanning electron microscopy. Among 47 Prorocentrum strains, seven morphospecies were identified as P. lima, P. rhathymum, P. concavum, P. cf. emarginatum, P. fukuyoi, P. cf. maculosum and P. panamense. The latter five species have not been previously reported in Chinese waters, and this is the first record of P. panamense outside its type locality. Partial large subunit (LSU) ribosomal DNA and internal transcribed spacer region sequences were obtained and molecular phylogenetic analysis was carried out using maximum likelihood and Bayesian inference. Chinese P. cf. maculosum strains share 99.5% similarity of LSU sequences with the strain from Cuba (close to the type locality), but Chinese P. lima strains share only 96.7% similarity of LSU sequences with the strain from the type locality. P. cf. emarginatum differs from P. fukuyoi mainly in the presence/absence of marginal pores and they form a well-resolved clade together with P. sculptile. OA was detected in all Chinese strains of P. lima and P. cf. maculosum based on liquid chromatography-mass spectrometry analysis, but dinophysistoxin was produced only by two P. lima strains. Chinese strains of P. concavum, P. rhathymum, and P. panamense do not produce detectable level of OA. Our results support the wide distribution of epibenthic Prorocentrum species and highlight the potential risk of DSP in the northern South China Sea

Radiomics signature as a new biomarker for preoperative prediction of neoadjuvant chemoradiotherapy response in locally advanced rectal cancer

PURPOSEWhether radiomics methods are useful in prediction of therapeutic response to neoadjuvant chemoradiotherapy (nCRT) is unclear. This study aimed to investigate multiple magnetic resonance imaging (MRI) sequence-based radiomics methods in evaluating therapeutic response to nCRT in patients with locally advanced rectal cancer (LARC).METHODSThis retrospective study enrolled patients with LARC (06/2014-08/2017) and divided them into nCRT-sensitive and nCRT-resistant groups according to postoperative tumor regression grading results. Radiomics features from preoperative MRI were extracted, followed by dimension reduction using the minimum redundancy maximum relevance filter. Three machine-learning classifiers and an ensemble classifier were used for therapeutic response prediction. Radiomics nomogram incorporating clinical parameters were constructed using logistic regression. The receiver operating characteristic (ROC), decision curves analysis (DCA) and calibration curves were also plotted to evaluate the prediction performance.RESULTSThe machine learning classifiers showed good prediction performance for therapeutic responses in LARC patients (n=189). The ROC curve showed satisfying performance (area under the curve [AUC], 0.830; specificity, 0.794; sensitivity, 0.815) in the validation group. The radiomics signature included 30 imaging features derived from axial T1-weighted imaging with contrast and sagittal T2-weighted imaging and exhibited good predictive power for nCRT. A radiomics nomogram integrating carcinoembryonic antigen levels and tumor diameter showed excellent performance with an AUC of 0.949 (95% confidence interval, 0.892–0.997; specificity, 0.909; sensitivity, 0.879) in the validation group. DCA confirmed the clinical usefulness of the nomogram model.CONCLUSIONThe radiomics method using multiple MRI sequences can be used to achieve individualized prediction of nCRT in patients with LARC before treatment

Sympatric occurrence of two Azadinium poporum ribotypes in the Eastern Mediterranean Sea

The marine dinoflagellate Azadinium poporum produce azaspiracids (AZA) and has been recorded widely in the world. However, information on its biogeography is still limited, especially in view of the fact that A. poporum comprises several genetically differentiated groups. A total of 18 strains of A. poporum were obtained from the Eastern Mediterranean area by incubating surface sediment collected from Ionian Sea of Greece. The morphology of these strains was examined with light microscopy and scanning electron microscopy. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from all cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences confirmed three ribotypes within A. poporum and revealed two subclades within ribotypes A and C. Greek strains of A. poporum ribotype A were nested within ribotype A2 together with strains from Western Mediterranean Sea and French Atlantic, and Greek strains of A. poporum ribotype C were nested within ribotype C2 together with a strain from the Gulf of Mexico. Growth experiments on four selected strains revealed that ribotypes A and C from Greece differed in their growth at higher temperatures, indicating that they are physiologically differentiated. Azaspiracid profiles were analyzed for 15 cultured A. poporum strains using LCMS/MS and demonstrate that the A. poporum ribotype A from Greece produce low level or no AZA and A. poporum ribotype C from Greece produces predominantly AZA-40 (9.6–30.2 fg cell−1) followed by AZA-2 (2.1–2.6 fg cell−1). The first record of AZA-40 producing A. poporum from the Mediterranean suggests that this species is a potential source for azaspiracid contaminations in shellfish from the Eastern Mediterranean Sea

Adding new pieces to the Azadinium (Dinophyceae) diversity and biogeography puzzle: Non-toxigenic Azadinium zhuanum sp. nov. from China, toxigenic A. poporum from the Mediterranean, and a non-toxigenic A. dalianense from the French Atlantic

The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44 fg cell−1. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea

Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum.

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera

Diversity and seasonal occurrence of <I>Skeletonema</I> (Bacillariophyta) species in Xiamen Harbour and surrounding seas, China

Skeletonema costatum like cells are recorded globally and often generate a huge biomass when blooming. In the present study we performed a taxonomic and geographic survey of the species diversity in Skeletonema along the Chinese coastline. We concentrated our efforts on Xiamen Harbour, where we followed the annual cycle of the generic diversity by collecting 51 water samples from 2006 to 2010. A total of 687 strains of Skeletonema were established and they were identified as S. ardens, S. costatum, S. dohrnii, S. grevillei, S. menzelii, and S. tropicum. S. dohrnii is a winter-spring species, whereas other species tend to appear in summer and autumn. S. costatum is the single species present throughout the year. To assess how the species overcome the period in which they are not detectable in the water column we also sampled the surface sediments and either isolated resting stages and incubated them into cultures, or we generated monoclonal strains by incubating the sediment and isolated whatever emerged from it. The above six species except S. grevillei, and S. pseudocostatum as well, were detected this way, implying that the species produce resting stages. Resting cells of S. costatum and S. tropicum were actually observed in the sediment.</p

Toxic dinoflagellate blooms of Gymnodinium catenatum and their cysts in Taiwan Strait and their relationship to global populations

Gymnodinium catenatum is able to produce paralytic shellfish toxins (PSTs) and was responsible for a massive bloom in the Taiwan Strait, East China Sea, in June 2017, which resulted in serious human poisoning and economic losses. To understand the origin of the bloom and determine the potential for blooms in subsequent years, water and sediment samples collected in the Taiwan Strait from 2016 to 2019 were analyzed for cells and cysts using light microscopy (LM) and/or quantitative polymerase chain reaction (qPCR). The morphology of both cells and cysts from the field and cultures was examined with LM and scanning electron microscopy (SEM). Large subunit (LSU) and/or internal transcribed spacer (ITS)-5.8S rRNA gene sequences were obtained in 13 isolates from bloom samples and five strains from cysts. In addition, cells of strains TIO523 and GCLY02 (from the Taiwan Strait and Yellow Sea of China, respectively) were subjected to growth experiments, and cysts from the field were used for germination experiments under various temperatures. Our strains shared identical LSU and ITS-5.8S rRNA gene sequences with those from other parts of the world, and therefore belonged to a global population. A low abundance of G. catenatum cells were detected during most of the sampling period, but a small bloom was encountered in Quanzhou on June 8, 2018. Few cysts were observed in 2016 but a marked increase was observed after the bloom in 2017, with a highest density of 689 cysts cm−3. Cysts germinated at temperatures between 14 and 23 °C with a final germination rate over 93%. Strains TIO523 and GCLY02 displayed growth at temperatures between 17 and 26 °C and 14 and 26 °C, respectively, with both strains displaying the highest growth rate of ca. 0.5 divisions d–1 at 23 °C. The PSTs of the three strains and cysts from the sediments were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). All strains were able to produce PSTs, which were dominated by N-sulfocarbamoyl C toxins (C1/2, 53.0–143.5 pg cell−1) and decarbamoyl gonyautoxins (dcGTX2/3, 26.7–52.1 pg cell−1), although they were not detected in cysts. However, hydroxybenzoyl (GC) toxins were detected in both cells and cysts. Our results suggested that the population in the Taiwan Strait belonged to a warm water ecotype and has a unique toxin profile. Our results also suggested that the persistence of cells in the water column may have initiated the bloom

Impacts of Mesoscale Eddies on Biogeochemical Variables in the Northwest Pacific

Mesoscale eddies play an important role in regulating biogeochemical cycles. However, the response of biogeochemical variables to cold and warm eddies has not been well elucidated, mainly due to most previous studies relying on remote sensing techniques and lacking in situ observations below the surface water. Here, we used hydrographic and biochemical data from one survey in the northwestern Pacific to document the vertical biogeochemical structure of one cold and two warm eddies. We first compared the changes of key variables in the eddy core relative to eddy outside, explained the role of key layers (the mixing depth, pycnocline, nutricline, euphotic) in causing these changes, and then analyzed the main environmental factors affecting chlorophyll a (Chla) and phytoplankton communities. Finally we focused on the response mechanisms of key biogeochemical variables to the cold and warm eddies. The results showed that biological variables (Chla, microphytoplankton, picophytoplankton), salinity, dissolved inorganic nitrogen (DIN), dissolved inorganic phosphate (DIP), and dissolved inorganic silicate (DSi) in the cold eddy core increased by 0.2–134%, while in the warm eddy core, they decreased by 0.2–70% relative to the eddy outside. The cold and warm eddies were able to force the deep chlorophyll maximum (DCM), which rose or fell with the pycnocline, nutricline and euphotic depth (Zeu) as a whole. Cold eddies with a raised thermocline could lead to about 20 m elevated DCM and enhanced phytoplankton biomass when the nutricline and thermocline were coincident. In contrast, warm eddies drove isopycnals downward, resulting in a 10–25 m drop in DCM and a decrease in nutrient and Chla concentrations at the center of the eddies. The significant difference in the vertical structure of the phytoplankton community between the center and the outside of the eddy might be explained by the direct influence of both nutrient concentrations and stoichiometry changes. The contribution of microphytoplankton to total biomass was much smaller than that of picophytoplankton in oligotrophic waters where the DIN:DIP and DSi:DIN ratios are significantly low. Compared to nutrients, photosynthetically active radiation (PAR) might not be the main factor controlling phytoplankton biomass and abundance attributed to Zeu being consistently deeper than the mixed depth (Zm), whereas it was likely to be the key limiting factor affecting the vertical distribution of the phytoplankton community

Morphology, phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemicompensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific

Impacts of Mesoscale Eddies on Biogeochemical Variables in the Northwest Pacific

Mesoscale eddies play an important role in regulating biogeochemical cycles. However, the response of biogeochemical variables to cold and warm eddies has not been well elucidated, mainly due to most previous studies relying on remote sensing techniques and lacking in situ observations below the surface water. Here, we used hydrographic and biochemical data from one survey in the northwestern Pacific to document the vertical biogeochemical structure of one cold and two warm eddies. We first compared the changes of key variables in the eddy core relative to eddy outside, explained the role of key layers (the mixing depth, pycnocline, nutricline, euphotic) in causing these changes, and then analyzed the main environmental factors affecting chlorophyll a (Chla) and phytoplankton communities. Finally we focused on the response mechanisms of key biogeochemical variables to the cold and warm eddies. The results showed that biological variables (Chla, microphytoplankton, picophytoplankton), salinity, dissolved inorganic nitrogen (DIN), dissolved inorganic phosphate (DIP), and dissolved inorganic silicate (DSi) in the cold eddy core increased by 0.2&ndash;134%, while in the warm eddy core, they decreased by 0.2&ndash;70% relative to the eddy outside. The cold and warm eddies were able to force the deep chlorophyll maximum (DCM), which rose or fell with the pycnocline, nutricline and euphotic depth (Zeu) as a whole. Cold eddies with a raised thermocline could lead to about 20 m elevated DCM and enhanced phytoplankton biomass when the nutricline and thermocline were coincident. In contrast, warm eddies drove isopycnals downward, resulting in a 10&ndash;25 m drop in DCM and a decrease in nutrient and Chla concentrations at the center of the eddies. The significant difference in the vertical structure of the phytoplankton community between the center and the outside of the eddy might be explained by the direct influence of both nutrient concentrations and stoichiometry changes. The contribution of microphytoplankton to total biomass was much smaller than that of picophytoplankton in oligotrophic waters where the DIN:DIP and DSi:DIN ratios are significantly low. Compared to nutrients, photosynthetically active radiation (PAR) might not be the main factor controlling phytoplankton biomass and abundance attributed to Zeu being consistently deeper than the mixed depth (Zm), whereas it was likely to be the key limiting factor affecting the vertical distribution of the phytoplankton community