Ying Zhong Tang

Sexual resting cyst production by the dinoflagellate Akashiwo sanguinea: a potential mechanism contributing to the ubiquitous distribution of a harmful alga

The dinoflagellate Akashiwo sanguinea is a well known, cosmopolitan harmful microalga that frequently forms harmful algal blooms (HABs) in marine estuaries from temperate to tropical waters, and has posed a severe threat to fish, shellfish, and sea birds. Therefore, it is important to understand the ecology of this species, particularly the mechanisms regulating its ubiquitous geographic distribution and frequent recurrence of. To date, the mechanisms regulating distribution and recurrence of HABs by this species have been poorly understood. While resting cyst production can play a central role in the geographic expansion and initiation of HABs, studies of the life cycle of this alga, including cyst production, have been lacking. Here, we demonstrate that A. sanguinea produces sexual resting cysts homothallically. We present evidence for cell pairs in sexual mating, biflagellated planozygote formation, and cysts of different morphologies, and we describe time series for germination of cysts to germlings with two longitudinal flagella, along with studies of possible factors affecting cyst production. Phylogenetic analysis of large sub‐unit rDNA sequences revealed a monophyly of this species and thus possibly a recent common ancestor for all global populations. The discovery of resting cyst production by A. sanguinea suggests its frequent recurrence of blooms and global distribution may have been facilitated by the natural and anthropogenic transport of resting cysts.

Inhibition of marine coastal bloom-forming phytoplankton by commercially cultivated Gracilaria lemaneiformis (Rhodophyta)

While harmful algal blooms (HABs) have become a threat to fisheries, environmental and public health, and economies worldwide, most of the approaches under investigations for prevention, control, and mitigation (PCM) of HABs are limited by high cost and possible secondary pollution. This study reports our laboratory and field experiments demonstrating effective growth-inhibiting effects of the widely cultivated seaweed Gracilaria lemaneiformis on phytoplankton possibly forming HABs. Laboratory experiments demonstrated significant growth-inhibiting effects of fresh G. lemaneiformis thalli in a dose-response manner on four of six test microalgae commonly observed in the seaweed cultivation area (Scrippsiella trochoidea, Prorocentrum micans, Skeletonema costatum, Dunaliella salina) and on the natural phytoplankton assemblage in seawater. Significant inhibiting effects were also observed from in situ bottle (1.0xa0L) and mesocosm (50 and 1000xa0L) experiments on the harmful alga Phaeocystis globosa during its blooms. Based on a large-scale field survey, the phytoplankton abundance in the areas with Gracilaria cultivation was demonstrated to be significantly lower than that in the areas without Gracilaria. Collectively, our results showed that large-scale cultivation of G. lemaneiformis is an effective approach to prevent HABs in coastal waters, as an added value to the economically viable industry of Gracilaria cultivation.

The ability of the red macroalga, Porphyra purpurea (Rhodophyceae) to inhibit the proliferation of seven common harmful microalgae

Harmful algal blooms (HABs) caused by microalgae have become a serious threat to public health, tourism, fisheries, ecosystems, and economies around the world. Recent studies have indicated that some macroalgae may inhibit the growth of HABs-forming microalgae. The red macroalgae, Porphyra spp. (nori), are the most valuable maricultured seaweed with a global annual production worth of over

Discovery of a resting stage in the harmful, brown‐tide‐causing pelagophyte, Aureoumbra lagunensis: a mechanism potentially facilitating recurrent blooms and geographic expansion

US 1.8 billion. Here, we report on the ability of Porphyra purpurea, indigenous to the US east coast, to inhibit the growth of seven common harmful algae, including the brown tide-forming pelagophyte Aureococcus anophagefferens; the raphidophyte Chattonella marina; the toxic diatom Pseudo-nitzschia multiseries; and the dinoflagellates Cochlodinium polykrikoides, Karlodinium veneficum, Karenia brevis, and Prorocentrum minimum. Controlled laboratory experiments with saturating nutrients and normal pH levels demonstrated that fresh thalli of P. purpurea added at environmentally realistic levels (mgxa0L−1) strongly inhibited the growth of the harmful algae in a manner dependent on dose of both the macro- and microalgae. Extract, in equivalent dosages, of both fresh and dried P. purpurea also exhibited the same or more significant effects, which demonstrated that P. purpurea produced stable allelochemicals that contributed toward the observed growth-inhibiting effects. Experi-mentally co-culturing of live P. purpurea thalli and A. anophagefferens with high or low nutrient levels during harmful brown tides consistently yielded significant reductions in cell densities. High levels of macroalgae (fresh thalli and the extract of fresh thalli) did not alter the growth or survival of larval fish (Cyprinodon variegatus; 1xa0week old). Therefore, our results, together with the well-known nutrient-removal capacity of seaweeds, allow us to conclude that P. purpurea, and likely other species of Porphyra, can be cultured as an ecologically safe, environmentally friendly, and economically viable measure to prevent, control, or mitigate microalgal HABs in coastal ecosystems, adding benefit to integrated multitrophic aquaculture systems.

Effects of temperature, salinity, and irradiance on the growth of harmful algal bloom species Phaeocystis globosa Scherffel (Prymnesiophyceae) isolated from the South China Sea

To date, the life stages of pelagophytes have been poorly described. This study describes the ability of Aureoumbra lagunensis to enter a resting stage in response to environmental stressors including high temperature, nutrient depletion, and darkness as well as their ability to revert from resting cells back to vegetative cells after exposure to optimal light, temperature, and nutrient conditions. Resting cells became round in shape and larger in size, filled with red accumulation bodies, had smaller and fewer plastids, more vacuolar space, contained lower concentrations of chl a and RNA, displayed reduced photosynthetic efficiency, and lower respiration rates relative to vegetative cells. Analysis of vegetative and resting cells using Raman microspectrometry indicated resting cells were enriched in sterols within red accumulation bodies and were depleted in pigments relative to vegetative cells. Upon reverting to vegetative cells, cells increased their chl a content, photosynthetic efficiency, respiration rate, and growth rate and lost accumulation bodies as they became smaller. The time required for resting cells to resume vegetative growth was proportional to both the duration and temperature of dark storage, possibly due to higher metabolic demands on stored energy (sterols) reserves during longer period of storage and/or storage at higher temperature (20°C vs. 10°C). Resting cells kept in the dark at 10°C for 7 months readily reverted back to vegetative cells when transferred to optimal conditions. Thus, the ability of Aureoumbra to form a resting stage likely enables them to form annual blooms within subtropic ecosystems, resist temperature extremes, and may facilitate geographic expansion via anthropogenic transport.

Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR

Blooms of Phaeocystis globosa have been frequently reported in Chinese coastal waters, causing serious damage to marine ecosystems. To better understand the ecological characteristics of P. globosa in Chinese coastal waters that facilitate its rapid expansion, the effects of temperature, salinity and irradiance on the growth of P. globosa from the South China Sea were examined in the laboratory. The saturating irradiance for the growth of P. globosa (Is) was 60 μmol/(m2∙s), which was lower than those of other harmful algal species (70–114 μmol/(m2∙s)). A moderate growth rate of 0.22/d was observed at 2 μmol/(m2∙s) (the minimum irradiance in the experiment), and photo-inhibition did not occur at 230 μmol/(m2∙s) (the maximum irradiance in the experiment). Exposed to 42 different combinations of temperatures (10–31°C) and salinities (10–40) under saturating irradiance, P. globosa exhibited its maximum specific growth rate of 0.80/d at the combinations of 24°C and 35, and 27°C and 40. The optimum growth rates (>0.80/d) were observed at temperatures ranging from 24 to 27°C and salinities from 35 to 40. While P. globosa was able to grow well at temperatures from 20°C to 31°C and salinities from 20 to 40, it could not grow at temperatures lower than 15°C or salinities lower than 15. Factorial analysis revealed that temperature and salinity has similar influences on the growth of this species. This strain of P. globosa not only prefers higher temperatures and higher salinity, but also possesses a flexible nutrient competing strategy, adapted to lower irradiance. Therefore, the P. globosa population from South China Sea should belong to a new ecotype. There is also a potentially high risk of blooms developing in this area throughout the year.

Transcriptomic Analyses of Scrippsiella trochoidea Reveals Processes Regulating Encystment and Dormancy in the Life Cycle of a Dinoflagellate, with a Particular Attention to the Role of Abscisic Acid

The accurate measurement of gene expression via quantitative real-time reverse transcription PCR (qRT-PCR) heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of qRT-PCR to the HAB-forming group. In this study, six candidate reference genes, 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-tubulin (TUA), β-tubulin (TUB), actin (ACT) and cytochrome oxidase subunit 1 (COX1), were evaluated for their expression stability with qRT-PCR and three statistical algorithms (GeNorm, NormFinder, and BestKeeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages (resting cysts), different growth stages, in darkness, exposed to abscisic acid (ABA) and exposed to temperature stress. The results indicated that TUA, 18S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle, particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of qRT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.

Cultivation of seaweed Gracilaria lemaneiformis enhanced biodiversity in a eukaryotic plankton community as revealed via metagenomic analyses

Due to the vital importance of resting cysts in the biology and ecology of many dinoflagellates, a transcriptomic investigation on Scrippsiella trochoidea was conducted with the aim to reveal the molecular processes and relevant functional genes regulating encystment and dormancy in dinoflagellates. We identified via RNA-seq 3,874 (out of 166,575) differentially expressed genes (DEGs) between resting cysts and vegetative cells; a pause of photosynthesis (confirmed via direct measurement of photosynthetic efficiency); an active catabolism including β-oxidation, glycolysis, glyoxylate pathway, and TCA in resting cysts (tested via measurements of respiration rate); 12 DEGs encoding meiotic recombination proteins and members of MEI2-like family potentially involved in sexual reproduction and encystment; elevated expressions in genes encoding enzymes responding to pathogens (chitin deacetylase) and ROS stress in cysts; and 134 unigenes specifically expressed in cysts. We paid particular attention to genes pertaining to phytohormone signaling and identified 4 key genes regulating abscisic acid (ABA) biosynthesis and catabolism, with further characterization based on their full-length cDNA obtained via RACE-PCR. The qPCR results demonstrated elevated biosynthesis and repressed catabolism of ABA during the courses of encystment and cyst dormancy, which was significantly enhanced by lower temperature (4 ± 1°C) and darkness. Direct measurements of ABA using UHPLC-MS/MS and ELISA in vegetative cells and cysts both fully supported qPCR results. These results collectively suggest a vital role of ABA in regulating encystment and maintenance of dormancy, akin to its function in seed dormancy of higher plants. Our results provided a critical advancement in understanding molecular processes in resting cysts of dinoflagellates.

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

Plankton diversity reflects the quality and health of waters and should be monitored as a critical feature of marine ecosystems. This study applied a pair of 28S rRNA gene‐specific primers and pyrosequencing to assess the effects of large‐scale cultivation of the seaweed Gracilaria lemaneiformis on the biodiversity of eukaryotic plankton community in the coastal water of Guangdong, China. With 1 million sequences (2,221 operational taxonomic units [OTUs]) obtained from 51 samples, we found that the biodiversity of eukaryotic plankton community was significantly higher in the seaweed cultivation area than that in the nearby control area as reflected in OTU richness, evenness (Shannon–Wiener index) and dominance (Simpson index) for total plankton community and its four subcategories when Gracilaria biomass reached the maximum, while no such a significant difference was observed before seaweed inoculation. Our laboratory experiment using an artificial phytoplankton community of nine species observed the same effects of Gracilaria exposure. Principal component analysis and principal coordinates analysis showed the plankton community structure in cultivation area markedly differed from the control area when Gracilaria biomass reached its maximum. Redundancy analysis showed that G. lemaneiformis was the critical factor in controlling the dynamics of eukaryotic plankton communities in the studied coastal ecosystem. Our results explicitly demonstrated G. lemaneiformis cultivation could enhance biodiversity of plankton community via allelopathy, which prevents one or several plankton species from blooming and consequently maintains a relatively higher biodiversity. Our study provided further support for using large‐scale G. lemaneiformis cultivation as an effective approach for improving costal ecosystem health.

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

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.