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Unravelling the structure-function variation of phycospheric microorganisms and its ecological correlation with harmful macroalgal blooms (HMBs) is a challenging research topic that remains unclear in the natural dynamic process of HMBs. During the world’s largest green tide bloom, causative macroalgae Ulva prolifera experienced dramatic changes in growth state and environmental conditions, providing ideal scenarios for this investment. Here, we assess the phycospheric physicochemical characteristics, the algal host’s biology, the phycospheric bacterial constitutive patterns, and the functional potential during the U. prolifera green tide. Our results indicated that (i) variation in the phycosphere nutrient structure was closely related to the growth state of U. prolifera; (ii) stochastic processes govern phycospheric bacterial assembly, and the contribution of deterministic processes to assembly varied among phycospheric seawater bacteria and epiphytic bacteria; (iii) phycospheric seawater bacteria and epiphytic bacteria exhibited significant heterogeneity variation patterns in community composition, structure, and metabolic potential; and (iv) phycospheric bacteria with carbon or nitrogen metabolic functions potentially influenced the nutrient utilization of U. prolifera. Furthermore, the keystone genera play a decisive role in the structure-function covariation of phycospheric bacterial communities. Our study reveals complex interactions and linkages among environment-algae-bacterial communities which existed in the macroalgal phycosphere and highlights the fact that phycospheric microorganisms are closely related to the fate of the HMBs represented by the green tide. IMPORTANCE Harmful macroalgal blooms represented by green tides have become a worldwide marine ecological problem. Unraveling the structure-function variation of phycospheric microorganisms and their ecological correlation with HMBs is challenging. This issue is still unclear in the natural dynamics of HMBs. Here, we revealed the complex interactions and linkages among environment-algae-bacterial communities in the phycosphere of the green macroalgae Ulva prolifera, which causes the world’s largest green tides. Our study provides new ideas to increase our understanding of the variation patterns of macroalgal phycospheric bacterial communities and the formation mechanisms and ecological effects of green tides and highlights the importance of phycospheric microorganisms as a robust tool to help understand the fate of HMBs.

Large-scale green tides in the Yellow Sea occurred for 13 consecutive years since 2007. The unusual co-occurrence of green tides and golden tides occurred in the Yellow Sea in 2017. The causative species are Ulva prolifera and/or Sargassum horneri. Previous studies on physiological response characteristics of U. prolifera and S. horneri are done in the laboratory mainly, and there is no in-situ comparative study in this regard. In this study, the in-situ physiological response characteristics of both species were measured. The results indicated that cyclic electron flow and antioxidant system play more important roles in protecting U. prolifera, while non-photochemical quenching is more important for adapting to the environment in S. horneri. U. prolifera has a stronger ability to utilize nutrients to rapidly increase its biomass under a suitable condition compared to S. horneri.

It has been a scientific consensus that Ulva prolifera green tide that break out annually between May and August in the recent decade off eastern coast of Yellow Sea. To investigate the ecological mechanism of the decline of U. prolifera green tide, we compared the physiological characteristics of U. prolifera samples collected in three stages of the green tide in Subei Shoal north Jiangsu, the initiation point, on May 10 (early, the initiation), July 10 (middle, the outbreak), and in Taipingjiao, Qingdao on August 10 (late, the decline). Compared to the early samples, the middle, and late samples showed a lower chlorophyll content and photosynthetic efficiency at a higher lipid peroxidation level. The middle and later samples had fewer chloroplasts in the cells of U. prolifera than those in the early samples. In addition, the cells of U. prolifera sampled in the late stage showed ruptured and deformed cell walls. Differences in physiological function were reflected in the growth rate. Both the middle and late U. prolifera samples presented negative growth rates. Correlation analysis results show that the rising temperature was mainly responsible for the local decline of U. prolifera green tide. The high light-intensity that could induce the photoinhibition was also an important factor for the decline. Therefore, U. prolifera population that remained in the Subei Shoal and those that northward drifted to Qingdao experienced the same decline processes: rising temperature and continuous high light conditions in the late phase of green tide reduced the photosynthetic capacity and destroyed the antioxidant system of the thallus, which lead to decrease of biomass. The only difference is that the temperature in the Subei Shoal rose earlier than that in Qingdao; thus, U. prolifera remaining in the Subei Shoal went disappeared first.

Marine macrophytes (seagrasses and macroalgae) and their epiphytic microorganisms play an important role in the ecological and biochemical processes of coastal oceans. However, simultaneous comparative studies on the biodiversity and functions of epiphytic bacteria and fungi associated with marine macrophytes have not been conducted. In this study, high-throughput sequencing technology was used to describe the epiphytic bacterial and fungal communities of 11 common macroalgae and 2 seagrasses from an intertidal zone of northern China and compare them with seawater communities. The results showed that Proteobacteria and Bacteroidota were the dominant bacterial phyla in marine macrophytes, whereas Ascomycota, Chytridiomycota, and Basidiomycota were the dominant fungal phyla. The alpha diversity of the bacterial and fungal communities in seagrasses was the highest of all macrophyte samples. This may have been related to their ability to recruit microorganisms from multiple sources. Host phylogeny may influence bacterial community structure, and geographical differences may influence fungal community structure. The FAPROTAX data indicated that C metabolic microbes were enriched in marine macrophytes, while the FUNGuild data indicated that undefined saprotroph, which participated in organic matter degradation, were also enriched in marine macrophytes. These findings provide a theoretical basis regarding the epiphytic microorganisms of macrophytes and may offer new insights to support the improved ecological restoration of seagrass and macroalgae beds.

Ulva prolifera green tide in the Yellow Sea of China is a typical cross-regional marine ecological disaster. We hypothesized that the complex interactions between U. prolifera and its associated bacterial communities possibly impact the formation and outbreak of green tide. To test this hypothesis, the U. prolifera-associated bacterial community changes in the entire migration area were investigated through field sampling and high-throughput sequencing. The results showed that (1) with the green tide migration, the richness and diversity increased for U. prolifera epiphytic bacterial communities, while they decreased for seawater bacterial communities in the phycosphere. (2) The richness, diversity, and community composition of U. prolifera-associated bacteria changed more dramatically in the 35.00°N sea area. (3) Potential interactions between bacteria and U. prolifera existed during the entire long-distance migration of green tide, and six bacterial functional groups (BFGs) were defined. Growth-regulating BFG I and antibacterial and stress-resistance BFG II were the dominant communities in the early stage of the green tide migration, which have the role of regulating algal growth and synergistic protection. Heterotrophic BFG III and algicidal BFG IV were the dominant communities in the late stage of the green tide migration, and they were able to compete with algae for nutrients and inhibit algal growth. Nutritive BFG V and algae-derived nutritional type BFG VI symbiotically lived with algal host. Our study highlights the spatial and temporal complexity of U. prolifera-associated bacterial communities and provides valuable insights into the potential contribution of U. prolifera-associated bacterial communities to green tide outbreaks.

The physiological characteristics of Ulva prolifera and Blidingia sp. during two pre-bloom stages (March & May) were compared to evaluate the competitive advantage of U. prolifera on Pyropia aquaculture rafts in Subei Shoal. (1) Compared to Blidingia sp., U. prolifera had a lower growth rate, chlorophyll content, photosynthetic efficiency, and antioxidant capacity in March. (2) In May, various indicators of U. prolifera's physiological function improved significantly, while the antioxidant capacity of Blidingia sp. decreased significantly. Large lipidic globules in U. prolifera cells became scattered small lipidic globules in May, which indicated a decrease in lipid membrane peroxidation. (3) In U. prolifera, the ratio of buoyancy to gravity of per unit volume was 1.73, and the bubbles inside the thalli provided 60% of the total buoyancy. Buoyancy generated by the inflatable structure of U. prolifera allowed this species to float after being separated from the rafts.

Ocean acidification in coastal seawaters is a complex process, with coastal pH being affected by numerous factors including watershed and biological processes that also support metabolically diverse bacterial communities. The world's largest macroalgal blooms have occurred consecutively in the Yellow Sea over the last 13 years. In particular, algal mats formed by Yellow Sea green tides (YSGT) significantly influence coastal environments. Herein, we hypothesized that 1) inorganic carbonate chemistry in coastal areas is altered by diel metabolism of these giant algal mats and that 2) bacterial community composition in diffusive boundary layers might be altered along diel cycles due to algal mat metabolism. In situ studies indicated that algal mat metabolism led to changes in diel pH and CO2 in affected seawaters. Such metabolic activities could intensify diel pH fluctuations in algal mat diffusive boundary layers, as noted by pH fluctuations of 0.22 ± 0.01 units, and pCO2 fluctuations of 214.62 ± 29.37 μatm per day. In contrast, pH fluctuations of 0.11 ± 0.02 units and pCO2 fluctuations of 79.02 ± 42.70 μatm were noted in unaffected areas. Furthermore, the bacterial community composition associated with diffusive algal boundary layers, including those of ambient bacteria and epiphytic bacteria, exhibited diel changes, while endophytic bacterial communities were relatively stable. Flavobacteriaceae were particularly highly abundant taxa in the ambient and epiphytic bacterial communities and exhibited increased abundances at night but sharp decreases in abundances during daytime. Flavobacteriaceae are heterotrophic taxa that could contribute to coastal area acidification at night due to the transformation of organic carbon to inorganic carbon. These results provide new insights to understand the variability in coastal ocean acidification via harmful algal blooms while providing a framework for evaluating the effects of YSGT on costal carbon cycling.

During yellow sea green tide outbreak, the thalli of Ulva prolifera drifted more than 350 km on the sea surface from offshore of Jiangsu Province to Qingdao city, where they were exposed to complex changes of local environments. The purpose of this study is to investigate the response of the thalli to diurnal changes of environments on the sea surface, and a sea surface environment simulator (SSES) was designed to simulate the natural environment of floating U. prolifera mat. A control experiment is designed in the laboratory, which was processed under suitable conditions (20°C for temperature, 72 μmol photons/(m 2 ·s) for light intensity and 30 for salinity), and an in-situ research was conducted to study the photosynthetic responses of floating U. prolifera to diurnal changes of environments. The results show that the photosynthetic efficiency of the thalli decreased gradually with time, and decreased rapidly after 14:00 local time (LT). After exposed to the environment on the sea surface for 6 h, the photosynthetic activity of the thalli decreased significantly. Furthermore, physiological-level and molecular-level experiments revealed that non-photochemical quenching (NPQ), cyclic electron flow (CEF) and energy redistribution between PSI and PSII all played an important role in the strong photosynthetic plasticity of U. prolifera. NPQ is the most important photoprotective responses to environmental changes before 12:00 LT. The results also confirmed that the CEF and energy redistribution between PSI and PSII are the main synergistic effects for the thalli to adapt to the environmental changes when the process NPQ cannot work. And the result can further reveal the reason why U. prolifera can adapt to the living condition of long distance drift on the sea surface. The findings of this research could provide a theoretical basis for explaining outbreaks of the green tide and instructing the management of the problem.

Ultraviolet-B (280−320 nm wavelength; UV-B) is one of the important reactive oxygen species (ROS) production factors in plants. The use of chlorofluorocarbons (CFCs) as refrigerants or propellants caused a serious depletion of ozone in the 1980s, which led to an increase of UV-B. Although the emissions of CFCs have decreased significantly in recent years, the ozone layer will not recover soon. The slow recovery of the ozone causes the slow decrease of UV-B intensity, and the intensity of UV-B varies with time and weather throughout the day. In the present study, typical intertidal macroalgae Ulva prolifera was exposed to moderate and extreme UV-B radiation (1 W m-2 and 5 W m-2), and the production and roles of ROS in U. prolifera under UV-B are discussed. The results are as follows: 1) ROS were mainly produced in the chloroplast under moderate UV-B radiation (1 W m-2). ROS played the signaling molecule function and led the secondary signaling pathways during this process. 2) Excessive ROS were produced in both the chloroplast and peroxisome. OH caused irreversible damage to the thalli exposed to 5 W m−2 radiation. Overall, these novel results support the idea of dual roles of ROS in U. prolifera under UV-B radiation (signaling molecules and oxidative damage). Moreover, they further highlight the role of photosynthesis in the production of ROS under UV-B.

For 8 consecutive years, a green tide has originated in the southern Yellow Sea and spread to the Qingdao offshore area. The causative species, Ulva prolifera, always forms a very thick thallus mat that is capable of drifting long distances over long periods. During this process, although the thalli face disturbance by complex environmental factors, they maintain high biomass and proliferation. We hypothesized that some form of photosynthetic adaptation strategy must exist to protect the thalli. Therefore, we studied the different photosynthetic response characteristics of the surface and lower layers of the floating thallus mats, and investigated the physiological and molecular-level adaptation mechanisms. The results showed that: (1) U. prolifera has strong photosynthetic capability that ensures it can gain sufficient energy to increase its biomass and adapt to long-distance migration. (2) Surface layer thalli adapt to the complex environment by dissipating excess energy via photosynthetic quantum control (energy quenching and energy redistribution between PSII/PSI) to avoid irreversible damage to the photosynthetic system. (3) Lower layer thalli increase their contents of Chlorophyll a (Chl a) and Chlorophyll b (Chl b) and decrease their Chl a/Chl b ratio to improve their ability to use light energy. (4) U. prolifera has strong photosynthetic plasticity and can adapt to frequent exchange between the surface and lower layer environments because of wave disturbance. Pigment component changes, energy quenching, and energy redistribution between PSII/PSI contribute to this photosynthetic plasticity.

Ulva prolifera green tide is a serious marine ecological problem in China’s coastal areas, with recurrent outbreaks occurring annually during late spring and summer since 2007. Marine bacteria communities are closely linked with important ecological functions in coastal environments. Hence, the succession of bacterial community structures in seawater and sediments during the outbreak and extinction stages of U. prolifera green tide were studied in this paper. The results revealed that: (1) the outbreak of a green tide led to changes in the bacterial community structure and a reduction in the abundance and diversity of the bacterial community in the Qingdao offshore region. This change was more significant in sediments than in seawater. (2) The bacterial assemblage appeared to be dominated by members of Deltaproteobacteria, Bacteroidia and Flavobacteria in sediment, and reductions in the relative abundance of Flavobacteria and Alphaproteobacteria were also observed in seawater after the green tide outbreak. There was also a high similarity in species composition of bacteria in sediment (extinction stage) and seawater (outbreak stage). (3) The outbreak of the U. prolifera affected the abundance of functional bacterial communities in the offshore environment such as SRB (sulfate-reducing bacteria), CFB (Cytophaga-Flavobacter-Bacteroides), heterotrophic bacteria and the potentially algicidal bacteria Alteromonadaceae. Overall, U. prolifera affects aquatic ecosystem secondary production, biogenic geochemical cycles and the growth of other seaweeds in Qingdao offshore areas by driving variations in functional bacterial communities.

Large-scale green tides have occurred continuously in the Yellow Sea of China from 2007 to 2018, and the causative species of the Yellow Sea green tide (YSGT) is Ulva prolifera. The thalli form floated thallus mats, and the thalli from different layers of the thallus mat suffer significantly different environmental conditions. In the present study, the environmental conditions of the surface layer (SL), middle layer (ML), and lower layer (LL) of the thallus mat from mid-June (Stage I) to mid-July (Stage II) were simulated. Photosynthetic traits and antioxidant systems were measured. The results showed that (1) photoprotective [non-photochemical quenching (NPQ) and cyclic electron transport (CEF)] and antioxidant systems both play important roles in protecting against abiotic factors in U. prolifera. (2) Cooperation between NPQ and CEF was observed in the ML group; CEF and the antioxidant system in the SL group work synergistically to protect the thalli. Furthermore, an inferred spatiotemporal attribute regarding the YSGT is presented: the significant changes in abiotic factors on the sea surface can easily affect the thalli of SL and ML from mid-June to mid-July, and those of LL can be affected in mid-July. This cooperation combined with the spatiotemporal attributes offers an explanation for the annual occurrence of the YSGT. HIGHLIGHTS –Adaptive mechanisms of Ulva prolifera against abiotic factors. –Cooperation between photosynthetic and antioxidant systems. –Spatiotemporal attributes regarding the Yellow Sea green tide are presented.

Green tides have occurred in the Yellow Sea, China, every year from 2007 to 2015. The free-floating Ulva prolifera (Muller) J. Agardh was the causative macroalgal species. The co-occurring, attached U. intestinalis was also observed. Photosynthetic capacities were determined using chlorophyll fluorescence in situ and after 7 days lab acclimation, and a significant differences were noted. Pigment composition showed no obvious differences, but concentrations varied significantly, especially chlorophyll b in U. prolifera two times increase was observed after acclimation. The optimal photochemical efficiency of PS II (Fv/Fm) was significantly higher in U. prolifera. Photosynthetic rate (α), maximum relative electron transport rate (rETRmax), and minimum saturating irradiance (Ek), obtained from rapid light response curves (RLCs), showed almost the same photosynthetic physiological status as Fv/Fm. Quenching coefficients and low temperature (77 K) chlorophyll fluorescence emission spectra of thylakoid membranes analysis showed U. prolifera has a better recovery activity and plasticity of PSII than U. intestinalis. Furthermore, energy dissipation via non-photochemical quenching (NPQ) and state transitions showed efficacious photoprotection solution especially in U. prolifera suffered from the severe stresses. Results in the present study suggested that U. prolifera’s higher photosynthetic capacity would contribute to its free-floating proliferation, and efficacious photoprotection in addition to favorable oceanographic conditions and high nutrient levels support its growth and aggregation.