DNA and RNA Stability of Marine Microalgae in Cold-Stored Sediments and Its Implications in Metabarcoding Analyses.

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection.

Multiple resistance of Echinochloa phyllopogon to synthetic auxin, ALS-, and ACCase-inhibiting herbicides in Northeast China.

Echinochloa phyllopogon is a self-pollinating allotetraploid weed and a serious threat to global rice production. One sensitive and three multiple-resistant populations collected from two provinces of Northeast China were used to analyze the mechanism of multiple resistance of E. phyllopogon to penoxsulam, metamifop, and quinclorac. Compared with the sensitive population LN12, LN1 showed higher resistance to these three herbicides; LN24 showed medium resistance to penoxsulam and metamifop and higher resistance to quinclorac (274-fold); HLJ4 showed low resistance to penoxsulam and high resistance to metamifop and quinclorac. Target sequence analysis showed no mutations in acetolactate synthase or acetyl-CoA carboxylase genes. In-vitro enzyme activity analysis showed that the activity of the target enzyme of multiple herbicide-resistant populations was similar to that of the sensitive population. The P450 inhibitor, malathion, noticeably increased the sensitivity of LN1, LN24, and HLJ4 to penoxsulam, LN1 to metamifop, and HLJ4 to quinclorac. Under all four treatments, the GSTs activities of resistant and sensitive populations showed an increasing trend from day 1 to day 5, but the sensitivity and activity of GSTs were higher in the multiple-resistant population than that in the sensitive population LN12. This study identified the development of multiple-resistant E. phyllopogon populations that pose a serious threat to rice production in rice fields in Northeast China, preliminarily confirming that multiple-resistance was likely due to non-target-site resistance mechanisms. These populations of E. phyllopogon are likely to be more difficult to control.

Risk assessment for resistance to fludioxonil in Corynespora cassiicola in Liaoning China.

Cucumber corynespora leaf spot, caused by Corynespora cassiicola, is the primary disease of cucumber leaves in greenhouses in China. Fludioxonil is a phenylpyrrole fungicide that inhibits C. cassiicola growth. We studied the sensitivity of 170 isolates of C. cassiicola to fludioxonil and evaluated resistance risk. All of the isolates were sensitive to fludioxonil. The EC50 values ranged from 0.082 to 0.539 µg/mL with a mean of 0.207 ± 0.0053 µg/mL. Laboratory-created mutants with a high resistance factor to fludioxonil were genetically stable after 10 transfers and showed positive cross-resistance to iprodione and procymidone but not to azoxystrobin, carbendazim, pydiflumetofen, and prochloraz. There was no significant difference in mycelial growth and temperature adaptation between the mutant s and the sensitive isolates, except for pathogenicity and sporulation. The resistant isolates accumulated less glycerol than their parental isolates and were more sensitive to osmotic stress. The histidine kinase activity of the sensitive isolates was significantly inhibited compared to that of the resistant mutants. Sequence alignment of the histidine kinase gene CCos revealed that the mutants RTL4, RXM5, and RFS102 had point mutations at different sites that resulted in amino acid changes at G934E, S739F, and A825P in the CCos protein. The mutant RFS102 had an alanine deletion at site 824. After fludioxonil treatment, CCos expression by RFS20 was significantly lower than that of the parental isolate. Our findings demonstrate that C. cassiicola exhibits moderate resistance to fludioxonil.

Occurrence and mechanism of target-site resistance to bensulfuron-methyl in Monochoria korsakowii from China.

Monochoria korsakowii is an increasingly significant threat to rice production across China, particularly in Liaoning province. Few studies have reported herbicide resistance in M. korsakowii, and resistance status and mechanisms are poorly understood. Here, thirty field populations of M. korsakowii were collected from 11 rice-growing regions of Liaoning, and 97% of populations had evolved resistance to bensulfuron-methyl (BM), with majority (24 of 28) showing high resistance levels (RI > 10). The first in-depth analysis of molecular features of AHAS1 and AHAS2 in BM-resistant populations showed that four Pro197 mutations (Pro197 to His, Ala, Leu or Ser) in AHAS1 and one mutation (Pro197Ser) in AHAS2 were identified. Notably, novel double Pro197Ser mutations co-occurred in both AHAS1 and AHAS2 in the most resistant line LN-20. Furthermore, resistant mutants were used to investigate the effect of Pro197 mutations on AHAS functionality, binding modes, gene expression and cross-resistance in M. korsakowii. All the detected Pro197 mutations considerably reduced in vitro AHAS sensitivity to BM by weakening hydrogen bonds and hydrophobic interactions in the predicted BM-AHAS complexes, especially the double Pro197Ser mutations. This novel resistance mutation combination slightly impacted the extractable AHAS activity, and increased the affinity and catalytic rate of pyruvate. Also, the AHAS expression level was significantly up-regulated. Moreover, all mutations provided resistance only to other sulfonylureas herbicides but not triazolopyrimidine or pyrimidinyl-benzoates herbicides. In conclusion, bensulfuron-methyl resistance in M. korsakowii was grim in Liaoning, China, and amino acid mutations on AHAS isozymes were the primary resistance mechanism. Double Pro197Ser mutations in both AHAS1 and AHAS2 confer higher herbicide resistance than single mutations in AHAS1. Thus, this work deepens our understanding of resistance status and mechanisms of M. korsakowii.

Sensitivity to 12 Fungicides and Resistance Mechanism to Trifloxystrobin, Carbendazim, and Succinate Dehydrogenase Inhibitors in Cucumber Corynespora Leaf Spot (Corynespora cassiicola).

Corynespora cassiicola is the causal agent of cucumber Corynespora leaf spot, which affects many economically important plant species. Chemical control of this disease is hampered by the common development of fungicide resistance. In this study, 100 isolates from Liaoning Province were collected, and their sensitivity to 12 fungicides was determined. All the isolates (100%) were resistant to trifloxystrobin and carbendazim, and 98% were resistant to fluopyram, boscalid, pydiflumetofen, isopyrazam, and fluxapyroxad. However, none were resistant to propiconazole, prochloraz, tebuconazole, difenoconazole, and fludioxonil. The Cytb gene of trifloxystrobin-resistant isolates encoded the G143A mutation, whereas the ß-tubulin gene of carbendazim-resistant isolates encoded the E198A and E198A and M163I mutations. Mutations in SdhB-I280V, SdhC-S73P, SdhC-H134R, SdhD-D95E, and SdhD-G109V were associated with resistance to the succinate dehydrogenase inhibitors (SDHIs). Trifloxystrobin, carbendazim, and fluopyram were barely effective on the resistant isolates, whereas fludioxonil and prochloraz were effective on the isolates that were resistant to the quinone outside inhibitors (QoIs), SDHIs, and benzimidazoles. Ultimately, this study demonstrates that fungicide resistance seriously threatens the effective control of Corynespora leaf spot.

Different Geographic Strains of Dinoflagellate Karlodinium veneficum Host Highly Diverse Fungal Community and Potentially Serve as Possible Niche for Colonization of Fungal Endophytes.

In numerous studies, researchers have explored the interactions between fungi and their hosting biota in terrestrial systems, while much less attention has been paid to the counterpart interactions in aquatic, and particularly marine, ecosystems. Despite the growing recognition of the potential functions of fungi in structuring phytoplankton communities, the current insights were mostly derived from phytoplankton hosts, such as diatoms, green microalgae, and cyanobacteria. Dinoflagellates are the second most abundant group of phytoplankton in coastal marine ecosystems, and they are notorious for causing harmful algal blooms (HABs). In this study, we used high-throughput amplicon sequencing to capture global snapshots of specific fungal assemblages associated with laboratory-cultured marine dinoflagellate. We investigated a total of 13 clonal cultures of the dinoflagellate Karlodinium veneficum that were previously isolated from 5 geographic origins and have been maintained in our laboratory from several months to more than 14 years. The total recovered fungal microbiome, which consisted of 349 ASVs (amplicon sequencing variants, sequences clustered at a 100% sequence identity), could be assigned to 4 phyla, 18 classes, 37 orders, 65 families, 97 genera, and 131 species. The fungal consortium displayed high diversity and was dominated by filamentous fungi and ascomycetous and basidiomycetous yeasts. A core set of three genera among all the detected fungi was constitutively present in the K. veneficum strains isolated from geographically distant regions, with the top two most abundant genera, Thyridium and Pseudeurotium, capable of using hydrocarbons as the sole or major source of carbon and energy. In addition, fungal taxa previously documented as endophytes in other hosts were also found in all tested strains of K. veneficum. Because host-endophyte interactions are highly variable and strongly case-dependent, these fungal taxa were not necessarily genuine endosymbionts of K. veneficum; instead, it raised the possibility that dinoflagellates could potentially serve as an alternative ecological niche for the colonization of fungal endophytes. Our findings lay the foundation for further investigations into the potential roles or functions of fungi in the regulation of the growth dynamics and HABs of marine dinoflagellates in the field.

Characterizing the Status of Energetic Metabolism of Dinoflagellate Resting Cysts under Mock Conditions of Marine Sediments via Physiological and Transcriptional Measurements.

Similar to the seeds of higher plants, resting cysts, a non-motile, benthic, and dormant stage in the life history of many dinoflagellate species, play vital roles via germination in the seasonal dynamics and particularly the initiation of harmful algal blooms (HABs) of dinoflagellates. It is thus crucial for resting cysts to balance between the energetic catabolism for viability maintenance and the energy preservation for germination during their dormancy. Despite this importance, studies on how resting cysts of dinoflagellates accomplish energetic metabolism in marine sediment have been virtually absent. In this study, using the cosmopolitan HABs-causing species Scrippsiella acuminata as a representative, we measured the transcriptional activity of the most efficient pathway of the energy catabolism tricarboxylic acid (TCA) cycle, cell viability (via neutral red staining), and the cellular ATP content of resting cysts under a set of mock conditions in marine sediments (e.g., 4 °C, darkness, and anoxia) for a maximum period of one year. Based on the correlation analyses among the expression levels of genes, cyst viability, and ATP content, we revealed that the TCA cycle was still a crucial pathway of energetic catabolism for resting cysts under aerobic conditions, and its expression was elevated at higher temperatures, light irradiation, and the early stage of dormancy. Under anaerobic conditions, however, the TCA cycle pathway ceased expression in resting cysts, as also supported by ATP measurements. Our results have laid a cornerstone for the comprehensive revelation of the energetic metabolism and biochemical processes of dormancy of resting cysts in marine sediments.

Expression Patterns of the Heat Shock Protein 90 (Hsp90) Gene Suggest Its Possible Involvement in Maintaining the Dormancy of Dinoflagellate Resting Cysts.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone functioning in cellular structural folding and conformational integrity maintenance and thus plays vital roles in a variety of biological processes. However, many aspects of these functions and processes remain to be fully elucidated, particularly for non-model organisms. Dinoflagellates are a group of eukaryotes that are exceedingly important in primary production and are responsible for the most harmful algal blooms (HABs) in aquatic ecosystems. The success of dinoflagellates in dominating the plankton community is undoubtedly pertinent to their remarkable adaptive strategies, characteristic of resting cyst production and broad tolerance to stresses of temperature and others. Therefore, this study was conducted to examine the putative roles of Hsp90 in the acclimation to temperature stress and life stage alterations of dinoflagellates. Firstly, we isolated the full-length cDNA of an Hsp90 gene (StHsp90) via RACE from the cosmopolitan HAB species Scrippsiella trochoidea and tracked its transcriptions in response to varied scenarios via real-time qPCR. The results indicated that StHsp90 displayed significant mRNA augment patterns, escalating during 180-min treatments, when the cells were exposed to elevated and lowered temperatures. Secondly, we observed prominently elevated StHsp90 transcriptions in the cysts that were stored at the cold and dark conditions compared to those in newly formed resting cysts and vegetative cells. Finally, and perhaps most importantly, we identified 29 entries of Hsp90-encoding genes with complete coding regions from a dinoflagellate-specific environmental cDNA library generated from marine sediment assemblages. The observed active transcription of these genes in sediment-buried resting cysts was fully supported by the qPCR results for the cold-stored resting cysts of S. trochoidea. Hsp90s expressions in both laboratory-raised and field-collected cysts collectively highlighted the possible involvement and engagement of Hsp90 chaperones in the resting stage persistence of dinoflagellates.

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the ß subunit of ATP synthase gene (ß-F1-ATPase), a key indicator for the status of cell's energetic metabolism. The full-length cDNA of ß-F1-ATPase gene from S.trochoidea (Stß-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stß-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stß-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stß-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments.

The toxic dinoflagellate Karenia mikimotoi has been well-known for causing large-scale and dense harmful algal blooms (HABs) in coastal waters worldwide and serious economic loss in aquaculture and fisheries and other adverse effects on marine ecosystems. Whether K. mikimotoi forms resting cysts has been a puzzling issue regarding to the mechanisms of bloom initiation and geographic expansion of this species. We provide morphological and molecular confirmation of sexually produced thin-walled resting cysts by K. mikimotoi based on observations of laboratory cultures and their direct detection in marine sediments. Light and scanning electron microscopy evidences for sexual reproduction include attraction and pairing of gametes, gamete fusion, formation of planozygote and thin-walled cyst, and the documentation of the thin-walled cyst germination processes. Evidence for cysts in marine sediments was in three aspects: positive PCR detection of cysts using species-specific primers in the DNA extracted from whole sediments; fluorescence in situ hybridization detection of cysts using FISH probes; and single-cell PCR sequencing for cysts positively labeled with FISH probes. The existence of sexually produced, thin-walled resting cysts by K. mikimotoi provides a possible mechanism accounting for the initiation of annually recurring blooms at certain regions and global expansion of the species during the past decades.

3,000 km and 1,500-year presence of Aureococcus anophagefferens reveals indigenous origin of brown tides in China.

The nonmotile, spherical, picoplanktonic (2-µm-sized) pelagophyte Aureococcus anophagefferens has caused numerous harmful blooms ("brown tides") across global marine ecosystems. Blooms have developed along the east coast of the USA since 1985, a limited number of times in South Africa around 1997, and frequently in China since 2009. As a consequence, the harmful blooms have caused massive losses in aquaculture and coastal ecosystems, particularly mortalities in cultured shellfish. Therefore, whether A. anophagefferens was recently introduced to China via natural/artificial transport of resting stage cells or has been an indigenous species has become a question of profound ecological significance and broad interest, which motivated our extensive investigation on the geographic and historical presence of this species in the seas of China. We applied a combined approach of extensive PCR-based detection and sequencing, germination experiments and monoclonal antibody staining of germlings to samples of surface sediment and sediment core (dated via combined isotopic measurements) collected from all four seas of China, and searched the supplementary data set of a recent Science publication. We discovered that A. anophagefferens does have a resting stage in the sediment, but it also has a wide geographic distribution both in China (covering a range of ~30° in latitude, ~15.7° in longitude and 2.5-3,456 m in water depth; temperate to tropical and coastal to open oceans) and in almost all oceans of the world and a historical presence of >1,500 years in the Bohai Sea, China. The work revealed that A. anophagefferens is not a recently introduced, but an indigenous species in China and has in fact a globally cosmopolitan distribution.

Cloning and Partial Characterization of a Cold Shock Domain-Containing Protein Gene from the Dinoflagellate Scrippsiella trochoidea.

CSPs, cold shock domain (CSD) containing proteins, are demonstrated to be involved in low temperature responses and various cellular processes under normal growth conditions. Here, we used the cosmopolitan, toxic, and resting cyst-producing dinoflagellate Scrippsiella trochoidea as a representative harmful algal bloom-forming dinoflagellate to investigate the expression patterns of CSP in vegetative cells in response to temperature shocks and in resting cysts, with an objective to probe the possible function of CSP in dinoflagellates. The full-length cDNA of a CSP gene from S. trochoidea (StCSP) was obtained which has a solely N-terminal CSD with conserved nucleic acids binding motifs. The qPCR results together indicated StCSP expression was not modulated by temperature at the transcriptional level and implied this gene may not be associated with temperature stress responses in S. trochoidea as the gene's name implies. However, we observed significantly higher StCSP transcripts in resting cysts (newly formed and maintained in dormancy for different periods of time) than that observed in vegetative cells (at exponential and stationary stages), indicating StCSP is actively expressed during dormancy of S. trochoidea. Taking together our recent transcriptomic work on S. trochoidea into consideration, we postulate that StCSP may play roles during encystment and cyst dormancy of the species.

A trehalose-6-phosphate synthase gene from Saccharina japonica (Laminariales, Phaeophyceae).

The full-length cDNA sequence of a trehalose-6-phosphate synthase gene from Saccharina japonica (designated as SjaTPS) (Accession: KC578568) was isolated based on homologous cloning and RACE-PCR. It was 4,127 bp, with 320 bp 5'-UTR, 21 bp 3'-UTR, and open reading frame (ORF) of 3,786 bp. The deduced 1,261 amino acids characterized with predicted molecular weight of 137.84 kDa and theoretical isoelectric point of 7.12. The SjaTPS had one N-terminal CBM20 (family 20 carbohydrate-binding module) domain, one TPS domain (trehalose-6-phosphate synthase) in the middle region and a single TPP (trehalose-6-phosphate phosphatase) domain near the C-terminus. Structural analysis suggested that the SjaTPS putatively functioned as trehalose-6-phosphate synthase, and might be related to laminaran metabolism in S. japonica. Homology analysis indicated that the SjaTPS shared 49-70 % similarities with the 13 known TPS sequences of other algae; only 55 % amino acid similarities were detected between SjaTPS and the previously reported TPS sequence of S. japonica (Accession: DQ666325). Phylogenetic analysis revealed close affinity between SjaTPS and TPS of brown alga Ectocarpus siliculosus (Accession: CBJ29609). Transcriptional analysis showed that desiccation greatly enhanced SjaTPS expression and the maximum appeared at 3 h, which was about 300-fold compared to that of the start, implied that SjaTPS was involved with drought adaption in kelp. In vitro expression of SjaTPS showed that one distinct band existed at ~115 kDa, and western blot detection proved that it was positive to the anti-His antibody with high specificity. Our results increased the knowledge of trehalose-6-phosphate synthase properties in S. japonica and also important for better understanding the role trehalose plays in kelp abiotic tolerance for adaption to the sublittoral habitats.

The resting cyst of dinoflagellate Scrippsiella acuminata host bacterial microbiomes with more diverse trophic strategies under conditions typically observed in marine sediments.

Variation in the condition of marine sediments provides selective preservation milieus, which act as a key determinant for the abundance and distribution of dinoflagellate resting cysts in natural sediments. Microbial degradation is an understudied biological factor of potential importance in the processes. However, gaps remain in our knowledge about the fundamental information of the bacterial consortia associated with dinoflagellate resting cysts both in laboratory cultures and in the field. Here we used Scrippsiella acuminata as a representative of cyst-producing dinoflagellates to delineate the diversity and composition of bacterial microbiomes co-existing with the laboratory-cultured resting cysts, and to explore possible impacts of low temperature, darkness, and anoxia (the mock conditions commonly observed in marine sediments) on the associated bacterial consortia. Bacterial microbiome with high diversity were revealed associated with S. acuminata at resting stage. The mock conditions could significantly shift bacterial community structure and exert notably inhibitory effects on growth-promoting bacteria. Resting cysts under conditions typically observed in marine sediments fostered bacterial microbiomes with more diverse trophic strategies, characteristic of prominently enriched anaerobic chemotrophic bacteria generating energy via respiration with several different terminal electron acceptors, which yielded more acidic milieu unfavorable for the preservation of calcareous resting cysts. Our findings suggest that there is complex and dynamic interaction between dinoflagellates resting cysts and the associated bacterial consortia in natural sediments. This intrinsic interaction may influence the maintenance and/or accumulation of dinoflagellate resting cysts with potential of germination and initiation blooms in the field.

Evidence for the production of asexual resting cysts in a free-living species of Symbiodiniaceae (Dinophyceae).

Coral reef ecosystems are the most productive and biodiverse marine ecosystems, with their productivity levels highly dependent on the symbiotic dinoflagellates belonging to the family Symbiodiniaceae. As a unique life history strategy, resting cyst production is of great significance in the ecology of many dinoflagellate species, those HABs-causing species in particular, however, there has been no confirmative evidence for the resting cyst production in any species of the family Symbiodiniaceae. Based on morphological and life history observations of cultures in the laboratory and morpho-molecular detections of cysts from the marine sediments via fluorescence in situ hybridization (FISH), cyst photography, and subsequent singe-cyst PCR sequencing, here we provide evidences for the asexual production of resting cysts by Effrenium voratum, the free-living, red tide-forming, and the type species of the genus Effrenium in Symbiodiniaceae. The evidences from the marine sediments were obtained through a sequential detections: Firstly, E. voratum amplicon sequence variants (ASVs) were detected in the cyst assemblages that were concentrated with the sodium polytungstate (SPT) method from the sediments collected from different regions of China Seas by high-throughput next generation sequencing (NGS); Secondly, the presence of E. voratum in the sediments was detected by PCR using the species-specific primers for the DNA directly extracted from sediment; Thirdly, E. voratum cysts were confirmed by a combined approach of FISH using the species-specific probes, light microscopic (LM) photography of the FISH-positive cysts, and a subsequent single-cyst PCR sequencing for the FISH-positive and photographed cysts. The evidences from the laboratory-reared clonal cultures of E. voratum include that: 1) numerous cysts formed in the two clonal cultures and exhibited a spherical shape, a smooth surface, absence of ornaments, and a large red accumulation body; 2) cysts could maintain morphologically intact for a storage of two weeks to six months at 4 °C in darkness and of which 76-92 % successfully germinated through an internal development processes within a time period of 3-21 days after being transferred back to the normal culturing conditions; 3) two or four germlings were released from each cyst through the cryptopylic archeopyle in all cysts with continuous observations of germination processes; and 4) while neither sexual mating of gametes nor planozygote (cells with two longitudinal flagella) were observed, the haploidy of cysts was proven with flow cytometric measurements and direct LM measurements of fluorescence from cells stained with either propidium iodide (PI) or DAPI, which together suggest that the cysts were formed asexually. All evidences led to a conclusion that E. voratum is capable of producing asexual resting cysts, although its sexuality cannot be completely excluded, which guarantees a more intensive investigation. This work fills a gap in the knowledge about the life cycle, particularly the potential of resting cyst formation, of the species in Symbiodiniaceae, a group of dinoflagellates having unique life forms and vital significance in the ecology of coral reefs, and may provide novel insights into understanding the recovery mechanisms of coral reefs destructed by the global climate change and suggest various forms of resting cysts in the cyst assemblages of dinoflagellates observed in the field sediments, including HABs-causing species.

Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Abundant Species Diversity and Essential Functions of Bacterial Communities Associated with Dinoflagellates as Revealed from Metabarcoding Sequencing for Laboratory-Raised Clonal Cultures.

Interactions between algae and bacteria represent an important inter-organism association in aquatic environments, which often have cascading bottom-up influences on ecosystem-scale processes. Despite the increasing recognition of linkages between bacterioplankton and dynamics of dinoflagellate blooms in the field, knowledge about the forms and functions of dinoflagellate-bacteria associations remains elusive, mainly due to the ephemeral and variable conditions in the field. In this study, we characterized the bacterial community associated with laboratory cultures of 144 harmful algal strains, including 130 dinoflagellates (covering all major taxonomic orders of dinoflagellates) and 14 non-dinoflagellates, via high-throughput sequencing for 16S rRNA gene amplicons. A total of 4577 features belonging to bacteria kingdom comprising of 24 phyla, 55 classes, 134 orders, 273 families, 716 genera, and 1104 species were recovered from the algal culture collection, and 3 phyla (Proteobacteria, Bacteroidetes, and Firmicutes) were universally present in all the culture samples. Bacterial communities in dinoflagellates cultures exhibited remarkable conservation across different algal strains, which were dominated by a relatively small number of taxa, most notably the γ-proteobacteria Methylophaga, Marinobacter and Alteromonas. Although the bacterial community composition between dinoflagellates and non-dinoflagellate groups did not show significant difference in general, dinoflagellates harbored a large number of unique features (up to 3811) with relatively low individual abundance and enriched in the potential methylotrophs Methylophaga. While the bacterial assemblages associated with thecate and athecate dinoflagellates displayed no general difference in species composition and functional groups, athecate dinoflagellates appeared to accommodate more aerobic cellulolytic members of Actinobacteria, implying a more possible reliance on cellulose utilization as energy source. The extensive co-occurrence discovered here implied that the relationships between these algal species and the bacterial consortia could be viewed as either bilaterally beneficial (i.e., mutualism) or unilaterally beneficial at least to one party but virtually harmless to the other party (i.e., commensalism), whereas both scenarios support a long-term and stable co-existence rather than an exclusion of one or the other. Our results demonstrated that dinoflagellates-associated bacterial communities were similar in composition, with enrichment of potential uncultured methylotrophs to one-carbon compounds. This work enriches the knowledge about the fundamental functions of bacteria consortia associated with the phycospheres of dinoflagellates and other HABs-forming microalgae.

Identification and implications of a core bacterial microbiome in 19 clonal cultures laboratory-reared for months to years of the cosmopolitan dinoflagellate Karlodinium veneficum.

Identification of a core microbiome (a group of taxa commonly present and consistently abundant in most samples of host populations) is important to capture the key microbes closely associated with a host population, as this process may potentially contribute to further revealing their spatial distribution, temporal stability, ecological influence, and even impacts on their host's functions and fitness. The naked dinoflagellate Karlodinium veneficum is a cosmopolitan and toxic species, which is also notorious in forming harmful algal blooms (HABs) and causing massive fish-kills. Here we reported the core microbiome tightly associated with 19 strains of K. veneficum that were originally isolated from 6 geographic locations along the coast of China and from an estuary of Chesapeake Bay, United States, and have been maintained in the laboratory for several months to over 14 years. Using high-throughput metabarcoding of the partial 16S rRNA gene amplicons, a total of 1,417 prokaryotic features were detected in the entire bacterial microbiome, which were assigned to 17 phyla, 35 classes, 90 orders, 273 families, and 716 genera. Although the bacterial communities associated with K. veneficum cultures displayed heterogeneity in feature (sequences clustered at 100% sequence similarity) composition among strains, a core set of 6 genera were found persistent in their phycospheres, which could contribute up to 74.54% of the whole bacterial microbiome. Three γ-proteobacteria members of the "core," namely, Alteromonas, Marinobacter, and Methylophaga, were the predominant core genera and made up 83.25% of the core bacterial microbiome. The other 3 core genera, Alcanivorax, Thalassospira, and Ponticoccus, are reported to preferably utilize hydrocarbons as sole or major source of carbon and energy, and two of which (Alcanivorax and Ponticoccus) are recognized as obligate hydrocarbonoclastic bacteria (OHCB). Since OHCB generally present in extremely low abundance in marine water and elevate their abundance mostly in petroleum-impacted water, our detection in K. veneficum cultures suggests that the occurrence of obligate and generalist hydrocarbon-degrading bacteria living with dinoflagellates may be more frequent in nature. Our work identified a core microbiome with stable association with the harmful alga K. veneficum and opened a window for further characterization of the physiological mechanisms and ecological implications for the dinoflagellate-bacteria association.

Possible functions of CobW domain-containing (CBWD) genes in dinoflagellates using Karlodinium veneficum as a representative.

Since > 91% of dinoflagellates are proven auxotrophs of vitamin B12 and the cobalamin synthetase W (CobW) is a key gene involved in vitamin B12 synthesis pathway, a number of CobW domain-containing (CBWD) genes in dinoflagellates (DinoCBWDs) were surprisedly found from our transcriptomic and meta-transcriptomic studies. A total of 88 DinoCBWD genes were identified from the genomes and transcriptomes of four dinoflagellates, with five being cloned for full-lengths and characterized using the cosmopolitan and ecologically-important dinoflagellates Karlodinium veneficum and Scrippsiella trochoidea (synonym of Scrippsiella acuminata). DinoCBWDs were verified being irrelevant to vitamin B12 biosynthesis due to their transcriptions irresponsive to vitamin B12 levels and their phylogenetic positions. A comprehensive phylogenetic analysis demonstrated 75 out of the 88 DinoCBWD genes identified belong to three subfamilies of COG0523 protein family, of which most prokaryotic members are reported to be metallochaperones and the eukaryotic members are ubiquitously found but mostly unknown for their functions. Our results from K. veneficum demonstrated DinoCBWDs are associated with metal homeostasis and other divergent functions, with four KvCBWDs involving in zinc homeostasis and KvCBWD1 likely functioning as Fe-type nitrile hydratase activator. In addition, conserved motif analysis revealed the structural foundation of KvCBWD proteins that are consistent with previously described CBWD proteins with GTPase activity and metal binding. Our results provide a stepping-stone toward better understanding the functions of DinoCBWDs and the COG0523 family.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation.

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood. In this study, we further explored the effect of N and P deficiency on the algal growth and resting cyst production in the cosmopolitan HABs-causing species Scrippsiella acuminata via refining the N and P concentration gradients. Further, we tracked the expression patterns of one CyclinB and one CDK1 genes of S. acuminata at different growth stages under three deficiency concentrations (1/1000 dilutions of N, P, and both N and P). The results suggest that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth instead of inducing cyst formation. We also observed the highest cyst production when S. acuminata was cultured in the f/2-Si medium that was a one-thousandth dilution of N and P (N∼ 0.882 µM; P∼ 0.0362 µM). Our results for the expressions of CyclinB and CDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. Although we are still not sure whether the changing expressions of the two genes did regulate the transition of life cycle (i.e. cyst formation), or happened as parallels to the expressions of other truly regulating genes, our observations are surely inspirational for further investigations on the genetic regulation of life cycle transition in dinoflagellates. Our work will provide clues to probe the physiological and molecular mechanisms underlying the nutrient deficiency-induced alternation between life cycle stages in dinoflagellates.