Proteomic response of Phaeocystis globosa to nitrogen limitation

Phaeocystis globosa is an important unicellular eukaryotic alga that can also form colonies.P.globosa can cause massive harmful algal blooms and plays an important role in the global carbon or sulfur cycling.Thus far,the ecophysiology of P.globosa has been investigated by numerous studies.However,the proteomic response of P.globosa to nitrogen depletion remains largely unknown.We compared four protein preparation methods of P.globosa for two-dimensional electrophoresis(2-DE)(Urea/Triton X-100 with trichloroacetic acid(TCA)/acetone precipitation;TCA/acetone precipitation;Radio Immuno Precipitation Assay(RIPA)with TCA/acetone precipitation;and Tris buffer).Results show that the combination of RIPA with TCA/acetone precipitation had a clear gel background and showed the best protein spot separation effect,based on which the proteomic response to nitrogen depletion was studied using 2-DE.In addition,we identified six differentially expressed proteins whose relative abundance increased or decreased more than 1.5-fold(P<0.05).Most proteins could not be identified,which might be attributed to the lack of genomic sequences of P.globosa.Under nitrogen limitation,replication protein-like,RNA ligase,and sn-glycerol-3-phosphate dehydrogenase were reduced,which may decrease the DNA replication level and ATP production in P.globosa cells.The increase of endonucleaseⅢand transcriptional regulator enzyme may affect the metabolic and antioxidant function of P.globosa cells and induce cell apoptosis.These findings provide a basis for further proteomic study of P.globosa and the optimization of protein preparation methods of marine microalgae.

Factors inducing morphological transition in harmful algal bloom-forming species Phaeocystis globosa with emphasis on predator chemical cue

The microalga Phaeocystis globosa is recognized as a harmful alga and also one of the few keystone phytoplankton genera that shape the structure and function of marine ecosystems.P.globosa possess a complex polymorphic life cycle,exhibiting phase alternation between free-living cells of approximately 3-6μm in diameter and gelatinous colonies(palmelloid stage)reaching several millimeters.The knowledge on the factors that induced the morphological transition of P.globosa in the last two decades was reviewed.Emphasis is given to infochemicals,an additional biological factor induced by predator,with the attempt to reveal a relevant mechanism of induced morphological defense.

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

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 ofP. globosa （Is） was 60 μmol/（m^2·s）, which was lower than those of other harmful algal species （70-114μmol/（m^2·s））. A moderate growth rate of 0.22/d was observed at 2 μmol/（m^2·s） （the minimum irradiance in the experiment）, and photo-inhibition did not occur at 230 μmol/（m^2·s） （the maximum irradiance in the experiment）. Exposed to 42 different combinations of temperatures （10- 31 ℃） and salinities （10-40） under saturating irradiance, P. globosa exhibited its maximum specific growth rate of 0.80/d at the combinations of 24℃ and 35, and 27℃ and 40. The optimum growth rates （〉0.80/d） were observed at temperatures ranging from 24 to 27℃ and salinities from 35 to 40. While P. globosa was able to grow well at temperatures from 20℃ to 31℃ and salinities from 20 to 40, it could not grow at temperatures lower than 15℃ or salinities lower than 15. Factorial analysis revealed that temperature and salinity has similar influences on the growth of this species. This strain ofP. 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.

Molecular identification of bloom-forming species Phaeocystis globosa (Pryninesiophyta) and its dispersal based on rDNA ITS sequence analysis

The colony-forming Phaeocystis species are causative agents of dense bloom occurrences incoastal waters worldwide. It is difficult to separate them because of the different morphologies associated with their colonial stages. In this study we applied molecular approaches to analyze the genetic variation of Phaeocystis globosa and Phaeocystis pouchetii from several geographic regions, and to assist in tracing the dispersal of bloom-forming Phaeocystis species in coastal waters of China. The sequences of the internal transcribed spacers (ITS1 and ITS2) of rDNA and the 5. 8S ribosomal RNA gene of Phaeocystis strains were determined. Sequence comparison shows that P.globosa was the most divergent to P. pouchetii, exhibiting sequence divergence higher than 0.08. However, lower genetic divergences existed between strains of P. globosa. The sequence comparison of the Phaeocystis rDNA ITS clearly shows that the species isolated from the southeast coast of China is identified as P. globosa rather than P. cf. pouchetii or other species. Furthermore, the significance of rDNA variation in distinct global populations of P. globosa suggested it might have had sufficient time to accumulate detectable mutations at the rDNA locus, supporting the hypothesis of ancient dispersal of P .globosa to many areas, meaning that P. globosa blooms in the coastal waters of China are endemic rather than a newly introduced species or a foreign source. Finally, based on the high divergent region of rDNA ITS, a pair of species-specific primers for P. globosa were designed, they could be useful to detect the presence of this species in mixed plankton assemblages or flagellate stages that are recognized with diffculties by means of conventional microscopy.

Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay(NPA-SH)

Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c...

Phaeocystis globosa Scherffel,a harmful microalga,and its production of dimethylsulfoniopropionate

The production of dimethylsulfoniopropionate （DMSP） and its cleavage products （DMS） are well studied in phytoplankton worldwide. However, less is known about their sources, distributions, and impacts in the coast of China. We examined the production of DMSP and DMS in Phaeocvstis globosa Scherffel and other benthic macroalgae from the South China coast in relation to environmental conditions. P. globosa was a harmful marine microalgal species and its bloom took place in the eutrophic waters along the South China Sea frequently. It also produced high content of DMSP at different growth stages, with the highest concentration usually observed in the stationary period. Moreover, the production of DMSP in P. globosa was significantly affected by salinity and temperature with the highest contents associated with high salinity （e.g. 40） and low temperature （e.g. 20℃）. In field benthic macroalgae, there was also a marked difference in the DMSP of various species or different samples of the same species. Chlorophyll a contents were also determined for each macroalgal species. The highest chlorophyll a （238.7 ng/g fresh weight） was recorded in Chlorophyta Ulva lactuca at Guishan Island （Zhuhai）, while the lowest value （1.5 ng/g fresh weight） was found in Rhodophyta Gracilaria tenuistipitata in Zhanjiang. Further correlation analysis indicated that there was no significant relationship between the content of DMSP and chl-a in macroalgae samples （P〉 0.05）. All the results suggested that the production of DMSP in marine algae was not only species- and stage-related, but also greatly affected by various environmental factors.

Negative effects of Phaeocystis globosa on microalgae

The potential allelopathic effects of the microalga,Phaeocystis globosa Scherffel,on three harmful bloom algae,Prorocentrum donghaiense Lu,Chattonella marina(Subrahmanyan) Hara et Chihara and Chattonella ovata Hara et Chihara were studied.The growth of C.marina and C.ovata was markedly reduced when the organisms were co-cultured with P.globosa or cultured in cell-free spent medium.Haemolytic extracts from P.globosa cells in the senescence phase had a similar inhibitory effect on the three harmful bloom algae.However,P.globosa had less influence on the brine shrimp,Artemia salina.These results indicate that P.globosa may have an allelopathic effect on microalgae,which would explain the superior competitive abilities of P.globosa.Because the addition of the haemolytic toxins from P.globosa had similar effects on algae as spent media,these compounds may be involved in the allelopathic action of P.globosa.

Phaeocystis globosa Bloom Monitoring: Based on P. globosa Induced Seawater Viscosity Modification Adjacent to a Nuclear Power Plant in Qinzhou Bay, China

The mucus produced by the outbreak of Phaeocystis globosa in the adjacent waters of the Fangchenggang Nuclear Power Plant(FCGNPP) in China has blocked the entrance of the cooling water filtration system of the FCGNPP, and posed a threat to the safe operation of the FCGNPP. At present, there is no related research on whether the changes in seawater viscosity could be used as a new method for monitoring and providing early warning of P. globosa tide. During a complete red tide cycle, the temporal and spatial changes in the hydrological conditions(temperature, salinity, dissolved oxygen), chlorophyll-a(Chl-a), composition and abundance of phytoplankton, number and size of P. globose colonies, concentration of transparent exopolymer particles(TEP) and the seawater viscosity were measured in this study. The results indicate that there was an extremely significant negative correlation between the physical seawater viscosity η_(T,S) and temperature, and the correlation coefficient reached-0.998. The biological seawater viscosity η_(Bio) was positively correlated with the number of P. globosa colonies, and the correlation coefficients is 0.747. Because the increase in phytoplankton abundance, especially during the outbreak of P. globosa and a large amount of mucus produced by the colonies could significantly increase seawater viscosity, we suggest that biological factors were the main reasons for the increase in seawater viscosity. The η_(Bio) was completely consistent with the occurrence process of P. globosa bloom and could be used as a valuable index for P. globosa bloom monitoring.

Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world.The morphological,physiological,and biochemical characteristics are different among geographic strains,which can not be distinguished with nuclear ribosomal DNA markers at present.Therefore,the genetic distance and phylogeographic relationships of nuclear 28S rDNA D1–D2 and ITS regions,and three chloroplast intergenic spacers(petN-trnS1,trnM1-psbA,and rbcS-rpl27)were analyzed and compared among 13 strains of P.globosa isolated from the Pacific Ocean and Atlantic Ocean in this study.In addition,the nucleotide polymorphisms of 28S rDNA D1–D2,ITS,and rbcS-rpl27 regions were evaluated in two P.globosa strains.The various levels of nucleotide polymorphism were in the nuclear 28S rDNA D1–D2 region and ITS region,but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer.A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer,which had the strongest distinction to geographic strains compared to those of 28S rDNA D1–D2 and ITS regions.In the phylogenetic tree of rbcS-rpl27 intergenic spacer,the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P.globosa,and then formed an independent clade,while the other Atlantic strains and all of Pacific strains joined up to build the other clade.It was implied that at least two genetically distant populations of P.globosa existed in the Atlantic coastal regions.This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P.globosa,and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P.globosa.

The mechanism of a new type of modified clay controlling Phaeocystis globosa growth

Phaeocystis globosa is a harmful algal bloom(HAB)species worldwide.Using modified clay(MC)to control HABs and to mitigate their adverse effects is currently a commonly used method in China.In this paper,the effects of oxidized composite modified clay(OXI-MC)on P.globosa were studied from different perspectives.The results show that the OXI-MC could effectively remove P.globosa and inhibit both the growth of residual algal cells and the formation of new colonies.The P.globosa algal biomass removal efficiencies after 3 h reached 90%at a dose of 0.1 g/L,and the number of colonies with different particle sizes was greatly reduced.Compared with those of the control,the superoxide dismutase(SOD)activity,catalase(CAT)activity,and malondialdehyde(MDA)content of the residual algae significantly increased,indicating that OXI-MC caused oxidative stress in the algal cells.In addition,we evaluated the effects of OXI-MC on the photosynthesis of residual microalgae and found that the maximal photochemical efficiency of photosystem II(PSII)under dark adaptation(F v/F m)and actual photochemical efficiency of PSII(ФPSII)decreased,severely damaging the photosynthesis efficiency,implying that OXI-MC effected the photosynthesis system of P.globosa.The results of this study clarify that OXI-MC could remove the most of algal cells and break up the colonies of P.globosa by collision,flocculation,and releasing active substances,as well as inhibit effectively the growth and colony formation of residual P.globosa by causing oxidative stress,reducing photosynthesis activity,accelerating the degradation of polysaccharides,and inhibiting the formation of colonies.

Differences Between Solitary Cells and Colonial Cells in the Heteromorphic Life Cycle of Phaeocystis globosa: Morphology, Physiology, and Transcriptome

Large-scale blooms of Phaeocystis globosa have caused serious damage to marine ecosystems in coastal waters of China.Phaeocystis blooms depend on the competitive advantage of their heteromorphic life history:colony formation has the benefit of resisting herbivory by zooplankton,and solitary cells can absorb nutrients rapidly.In order to better understand the mechanisms underlying the metabolic differences between the two types of cells,morphological observations,rapid light curve analysis,fatty acid profiling,and transcriptome assessment were conducted in the laboratory.The rapid light curve of colonial cells was higher than that of solitary cells,which indicated that colonial cells had higher CO2 fixation capacity.The fatty acid level of colonial cells was evidently lower than that of solitary cells,which is consistent with down-regulated synthesis of fatty acids and up-regulated degradation of fatty acids in the transcriptome.ATP-binding cassette transporters,the TCA cycle,and biosynthesis of exopolysaccharides(EPS)also displayed obvious differences.In summary,colonial cells have stronger carbon fixation capacity.They do not synthesize fatty acids as energy storage materials but secrete EPS,which might be one of the mechanisms of colony formation.Here we present a physiological and molecular overview of the differences between solitary cells and colonial cells and thereby provide further insight to help unravel the mechanisms that help Phaeocystis globosa adapt to different environments.

Dynamics of Phaeocystis globosa bloom and implications for its seed sources in the Beibu Gulf,China

Algal blooms of haptophyte Phaeocystis globosa have been recorded in the Beibu Gulf,South China Sea(SCS)since 2011.Recent evidence suggests that TypeⅠand TypeⅣare the two dominant genotypes of P.globosa coexisting in this area,and their marker pigment profiles,colony sizes,and genetic information differ.Two genotype-specific quantitative polymerase chain reaction(qPCR)assays were used to clarify the distribution and dynamics of P.globosa bloom in the Beibu Gulffrom November 2018 to February 2019.Results show that the patterns of spatial-temporal distribution of TypeⅠand TypeⅣwere similar,the abundance of TypeⅣwas higher than that of TypeⅠduring the algal bloom,and thus TypeⅣcells dominated the P.globosa bloom in winter 2018.The relationships between environmental factors and P.globosa abundance indicated that temperature and nitrate were critical to the distribution and dynamics of P.globosa blooms in this area.The distribution of P.globosa cells in different water masses before the bloom revealed that the westward water via the Qiongzhou Strait,the SCS bottom water,and the current in the northwestern waters of Hainan Island could transport P.globosa cells into the Beibu Gulf serving as the seed sources triggering the intense blooms in the Beibu Gulf.

Stress regulation of photosynthetic system of Phaeocystis globosa and their hemolytic activity

Blooms of Phaeocystis globosa have been reported accountable for massive fi sh mortality worldwide.The toxigenic mechanisms of P.globosa,however,remain largely unclear due to the multiple structures and/or synergistic or antagonistic ef fects of hemolytic compounds.External stressors could lead to the regulation of photoprotective or antioxidative defense system,as well as the potential hemolytic activity.Therefore,the light-induced photosynthetic system,including the accessory photosynthetic growth,the relative electron transfer rate(ETR),photosynthetic e ffi ciency(F_(v)/F_(m)),quantum yield of photosystem II(Yield),together with the hemolytic activity of P.globosa were investigated under variable environmental conditions in the present study.Results confirmed that hemolytic activity of P.globosa was initiated by the light,but inhibited by low temperature(16℃),high light intensity(>100μmol/(m^(2)·s)),and iron-limited conditions.Interestingly,the hemolytic activity was not impacted by photosynthetic electron inhibitors(Diuron,atrazine,paraquat,and dibromothymoquinone),which signifi cantly inhibited the photosynthetic activity of P.globosa.The correlated response of hemolytic and photosynthetic activity of P.globosa under those environmental factors suggested that the hemolytic compounds of P.globosa would be involved in the photosynthetic process but not in the electron transfer chain of P.globosa.

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

: Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract of P. globosa are analyzed further. The effects of temperature, pH, different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37 °C, and at pH 7.0; Hg2+ was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg2+ either combines with the hemolysin or closes the pores, hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.

Isolation of toxic compounds from wild Phaeocystis globosa

Two new compounds, namely taenialactam C and globorin A（1 and 2）, as well as six known compounds,cornoside（3）, 2-phenylethyl-b-D-glucoside（4）, 3-isopropyl-5-acetoxycyclohexene-2-one-1（5）, 4-methyl-phenol（6）, 5-[（2S）-2-aminobutyl]-2-methyl-phenol（7）, and 1-（4-methylphenyl）-1-propanone（8） were isolated from wild Phaeocystis globosa. The structures of the new compounds were established by detailed spectroscopic analysis and by comparison with spectral data of related known compounds.The structures of the known compounds were identified by comparing their spectroscopic data with those reported in the literature. This paper also reports toxicity properties of the eight compounds against the brine shrimp Artemia salina and juvenile Epinephelus akaara fish. Some of these compounds showed significant lethality on the brine shrimp A. salina and the juvenile E. akaara fish.