Effects of Ocean Acidification on Nitrogen Metabolism of Skeletonema costatum

Ocean acidification(OA),caused by the rising concentration of atmospheric CO_(2),leads to changes in the marine carbonate system.This,in turn,affects the physiological processes of phytoplankton.In response to increased pCO_(2) levels,marine microalgae modulate their physiological responses to meet their energy and metabolic requirements.Nitrogen metabolism is a critical metabolic pathway,directly affecting the growth and reproductive capacity of marine microorganisms.Understanding the molecular mechanisms that regulate nitrogen metabolism in microalgae under OA conditions is therefore crucial.This study aimed to investi-gate how OA affects the expression profiles of key genes in the nitrogen metabolic pathway of the marine diatom Skeletonema costatum.Our findings indicate that OA upregulates key genes involved in the nitrogen metabolic pathway,specifically those related to nitrate assimilation and glutamate metabolism.Moreover,pCO_(2) has been identified as the predominant factor affecting the expression of these genes,with a more significant impact than pH variations in S.costatum.This research not only advances our understanding of the adaptive mechanisms of S.costatum in response to OA but also provides essential data for predicting the ecological consequences of OA on marine diatoms.

Morphological comparison and molecular marker screening of three Skeletonema species found in Changjiang(Yangtze)River Basin

In our recent investigations of diatom diversity,we studied three species,namely,Skeletonema costatum,Skeletonema subsalsum,and Skeletonema potamos.Although they have been found frequently in Changjiang(Yangtze)River Basin,their morphological and molecular identification is difficult in taxonomy.Therefore,to integrate morphological and molecular biological approaches,we compared systematically their morphological characters and performed phylogenetic analysis.Twelve strains of Skeletonema were collected and isolated from Shanghai and Jiangsu,China,and their morphological characteristics were examined by light microscopy(LM)and the scanning electron microscopy(SEM).Based on morphological comparison,we determined that S.potamos is easy to distinguish from the other two species.The heavily silicified areolae,undulated or cleft distal ends of terminal fultoportula processes(TFPPs),absence of basal pores of fultoportula processes(FPPs),the rootlike protrusions of FPPs,and no interlocking connection are the stable characteristics that can be used to identify S.potamos.However,there are only two features that can distinguish S.costatum from S.subsalsum,namely the location of terminal rimoportulae(TRPs)and the distal shape of TFPPs.In addition,we amplified and sequenced nine common genetic markers from the strains,from which 101 sequences were obtained,constructed phylogenetic trees based on the nine genes and evaluated that seven genes can be used to identify S.potamos,and revealed that S.subsalsum is the closest known relative of S.costatum,and only ATP synthetase beta-subunit gene(atp B)is able to distinguish them from each other,which strongly support that it is an effective molecular marker for Skeletonema.This work provided a theoretical basis for the taxonomic study of Skeletonema.

光照与东海近海中肋骨条藻(Skeletonema costatum)赤潮发生季节的关系

利用船基现场培养实验和模型计算的方法,研究了光照与东海近海春夏季均能发生中肋骨条藻赤潮的关系.结果表明,中肋骨条藻生长的最适光照强度(Iopt)随水温(t)的增加呈'慢升快降'的不对称'倒V'形变化特征,在25℃左右Iopt最大,为121.6 W.m-2,Iopt-t曲线符合Blanchard方程;由东海海表水温计算的中肋骨条藻Iopt的季节变化特征与东海海面光照强度、海水透明度的季节变化基本同步(R2=0.907±0.115,p<0.001),且滞后于海面光照强度2个月左右,这使春夏季海水中光照最适宜生长的水层均为全年最厚(6 m左右).可见,中肋骨条藻对光照的适应性很强,海区海水光照的适宜性是春夏季均能形成大规模中肋骨条藻赤潮的重要原因之一.

赤潮水体中胶体物质对赤潮异弯藻(Heterosigma akashiwo)和中肋骨条藻(Skeletonema costatum)生长的影响

2004年2月9~10日在胶州湾发生的柔弱几内亚藻(Guinardia delicatula)赤潮期间分别在3个典型赤潮站位采集表层水样,利用过滤(0.7μm)和切向超滤(1000Daltons)分离赤潮暴发水体中的胶体物质,分析了不同滤液及截留液的有机碳含量,并将赤潮异弯藻(Heterosigma akashiwo)和中肋骨条藻(Skeletonema costatum)在<1000Daltons的超滤液及具有不同含量胶体物质(1000Daltons^0.7μm)的截留液中进行培养。结果发现在发生柔弱几内亚藻赤潮期间水体中的溶解有机碳浓度会增加,特别是胶体有机碳浓度会成倍增长;在不同胶体含量海水中培养的赤潮异弯藻的最大生长率μmax和最大生物量Bf与对照组相比会随着胶体含量的增加而显著增大,而中肋骨条藻的最大生长率μmax和最大生物量Bf与对照组相比会随着胶体含量增加而显著降低,表明柔弱几内亚藻赤潮水体中的胶体物质对赤潮异弯藻的生长起促进作用,而对中肋骨条藻的生长起抑制作用。这说明柔弱几内亚藻在增殖过程中所产生的胶体物质具有他感作用,会影响其它微藻的生长,从而可能对柔弱几内亚藻赤潮的形成起重要作用。

粤东柘林湾中肋骨条藻(Skeletonema costatum)种群生态学

于2000年5月～2004年12月对粤东大规模养殖区柘林湾的赤潮藻中肋骨条藻（Skeletonema costatum）种群的时空分布进行了长达5a的调查。结果表明，中肋骨条藻种群密度的周年变动模式基本为双峰型，平面分布没有显著的空间差异。调查期间，中肋骨条藻种群密度的站位实测值为0～1．4×10^7cells／dm^3，总均值为3．3×10^5cells／dm^3，占浮游植物总细胞数的67．1％，为调查海区第1优势种。在总共1045份样品中，有中肋骨条藻出现的样品数为1020份。其中，种群密度大于10^6cells／dm^3的样品有65份，大于10^7cells／dm。则有4份。以大于10^6cells／dm^3为中肋骨条藻的赤潮密度标准，在调查期间至少于2000年、2003年发生4次赤潮。运用灰关联理论对中肋骨条藻种群密度与13个环境因子的关系进行排序分析发现，水温、pH值和浮游动物是影响柘林湾中肋骨条藻种群时空分布的关键因子。水温还与中肋骨条藻种群密度的对数值具极显著意义的线性关系，而达到赤潮密度的样品均落在24．5～32．0℃区间，即每年的5～9月份高温季节。由于柘林湾浮游动物的年高峰期也出现在高温季节，说明浮游动物摄食压力的存在可能是柘林湾中肋骨条藻赤潮发生的重要抑制因子。2004年调查海区中肋骨条藻种群密度和在浮游植物群落中的优势度骤然降低，可能与水体营养盐结构和Fe含量的变化有关。因此，长期调查与监测对于研究海湾生态学和赤潮发生机制是极为重要的。

塔玛亚历山大藻(Alexandrium tamarense)和中肋骨条藻(Skeletonema costatum)种间竞争及UV-B辐射胁迫对其影响

采用共培养的方法,研究了塔玛亚历山大藻和中肋骨条藻种群竞争关系对UV-B辐射增强的响应变化。结果表明,不同的接种密度对塔玛亚历山大藻和中肋骨条藻种群竞争有明显的影响。当塔玛亚历山大藻和中肋骨条藻的接种比例为A∶S=1∶4时,中肋骨条藻在竞争中占优势;当接种例为A∶S=1∶1时,虽然塔玛亚历山大藻在竞争中占优势,但也受到了中肋骨条藻的抑制;接种比例为A∶S=4∶1时,塔玛亚历山大藻的生长竞争优势更加明显。UV-B辐射增强(在预备实验的前提下,选择1.4J/m2)可改变塔玛亚历山大藻和中肋骨条藻种群竞争的关系,使塔玛亚历山大藻种群竞争能力降低,中肋骨条藻种群竞争能力大大提高,从而导致处理Ⅰ(A∶S=1∶4)中,中肋骨条藻在竞争优势更加明显,在处理Ⅱ(A∶S=1∶1)中,中肋骨条藻在竞争中占一定的优势。

The effect of UV-B radiation enhancement on the interspecific competition between Skeletonema costatum and Heterosigma akashiwo

The responses of the interspecific competition between Skeletonema costatum and Heterosigma akashiwo to UV-B radiation enhancement were studied by the co-culture method. The results showed that Heterosigma akashiwo exhibited inhibition on the growth of Skeletonema costatum, and with the increase of initial inoculation density of Heterosigma akashiwo, heavier inhibition on Skeletonema costatum appeared. Under different inoculation proportions, Heterosigma akashiwo could always be in predominance in competition with Skeletonema costatum. The UV-B radiation treatment could change the competition relationship between Skeletonema costatum and Heterosigma akashiwo, which could increase the competitive dominance of Skeletonema costatum and decrease the competitive dominance of Heterosigma akashiwo. When the inoculation proportions of Heterosigma akashiwo and Skeletonema costatum were H∶S=1∶4 and H∶S=1∶1,Skeletonema costatum was in predominance in this competition; however, Heterosigma akashiwo was in predominance when the inoculation proportion was H∶S=4∶1.

COMBINED EFFECTS OF TEMPERATURE, IRRADIANCE AND SALINITY ON GROWTH OF DIATOM SKELETONEMA COSTATUM

factor experiment was used to study the combined effects of temperature, irradiance and salinity on the growth of an HAB species diatom Skeletonema costatum (Grev.) Cleve. The results showed that temperature (12, 19, 25, 32℃), irradiance ((0.02, 0.08, 0.3, 1.6)×10 16 quanta/(s·cm 2)) and salinity (10, 18, 25, 30, 35) significantly influenced the growth of this species. There were interactive effects between any two of and among all three physical factors on the growth. In the experiment, the most optimal growth condition for S. costatum was temparature of 25℃, salinity of 18-35 and irradiance of 1.6×10 16 quanta/(s·cm 2). The results indicated S. costatum could divide at higher rate and were more likely to bloom under high temperature and high illumination from spring to fall. It was able to distribute widely in ocean and estuary due to its adaptation to a wide range of salinities.

Effects of modified clay flocculation on major nutrients and diatom aggregation during Skeletonema costatum blooms in the laboratory

Harmful algal blooms(HABs) can elicit several negative effects on aquatic environment(such as depleting the oxygen, blocking the sunlight, destroying the habitats of organisms) and life health(including poisoning/killing marine mammals, birds and human). Among the various control strategies for HABs(physical manipulation needs lots of manpower and expensive equipment, chemicals treatment has some toxic byproduct and high residual, microbial agents only has limited in laboratory research), the coagulation-flocculation of HAB species by modified clay(MC) has been proven to be an effective, lowcost and environmentally friendly method that has been widely applied in the field, particularly in eastern Asia. In order to examine the long-term effects of MC treatment, this study investigated the alternations in seawater of Skeletonema costatum, a high biomass dominant HAB species along the Chinese coast, by comparing the degradation of S. costatum detritus(A1) with the application of MC treatment(A2) and MC treatment in sediment condition(A3). The low dosage of 0.25 g/L MC could efficiently remove 4×108 cells/L of S. costatum cells within 3.5 h(approximately 97% removal). In addition, the results showed that both inorganic and organic nutrients were effectively reduced from seawater by MC particles. Compared to the total nitrogen(TN) and total phosphorus(TP) concentrations in A1 seawater, 44% of TN and 93% of TP in A2 seawater, as well as 72% of TN and 93% of TP in A3 seawater were removed during the onemonth incubation period. Simultaneously, 64% of DISi in A2 and 44% of DISi in A3 significantly decreased( P <0.001). This study demonstrated that MC treatment was able to significantly increase the downward flux of nutrients and delay the release velocity of inorganic nutrient from MC-algae matrix into the overlying seawater, particularly within sediment environment.

Allelopathic interactions between Prorocentrum micans and Skeletonema costatum or Karenia mikimotoi in laboratory cultures

Algal allelopathy is an ecological/physiological phenomenon that has focused attention on the interactions among algae and the production of algal toxins. We investigated the allelopathic interactions between the dinoflagellate genus Prorocentrum micans and diatom genus Skeletonema costatum and between P. micans and dinoflagellate genus Karenia mikimotoi using bi-algal cultures. Because the effects were species-specific and size-dependent, we evaluated the effect of different initial densities. At low densities of P. mieans and high densities of S. costatum inoculated into the same medium, the growth of R rnieans was weakly restrained, whereas the growth of S. costatum was significantly suppressed. S. costatum and K. mikimotoi were strongly inhibited by P. micans, in both the bi-algal cultures and enriched filtrates. Direct cell-to-cell contact was not necessary to gain a competitive advantage, thus, our results suggest that P. micans inhibited the growth of S. costatum and K. mikimotoi by the release of allelochemical（s）. Last, a mathematical model was used to simulate growth and interactions between P. micans and S. eostatum and between P. micans and K. mikimotoi in bi-algal cultures.

Influence of salinity and nitrogen content on production of dimethylsulfoniopropionate(DMSP)and dimethylsulfide(DMS)by Skeletonema costatum

The effects of changing salinity and nitrogen limitation on dimethylsulfoniopropionate(DMSP) and dimethylsulfide(DMS) concentrations were investigated in batch cultures of coastal diatom Skeletonema costatum,an ecologically important species.Changes in salinity from 20-32 caused no measurable variation in cell growth or culture yield,but increased intracellular DMSP per cell by 30%.Nitrogen limitation caused up to a two-fold increase in total DMSP per cell and up to a three-fold increase in DMS per cell.These changes in DMSP and DMS per cell in the Skeletonema costatum cultures with nitrogen limitation and changing salinity were primarily attributed to the physiological functions of DMSP as an osmolyte and an antioxidant.The data obtained in this study indicated that nitrogen limitation and salinity may play an important role in climate feedback mechanisms involving biologically derived DMS.

长江口骨条藻属(Skeletonema)种类多样性及其形态(英文)

利用光镜和扫描电镜(SEM)观察对长江口骨条藻属种类进行观察,共发现两种已有种:中肋骨条藻Skeletonema costatum(Greville)Cleve,玛氏骨条藻Skeletonema marinoi Sarno et Zingone.中肋骨条藻细胞直径为6.3～15.1μm,贯壳轴长度为7.7～11μm;玛氏骨条藻细胞直径为4.6～11.2μm,贯壳轴长度为11.7～12.2μm.中肋骨条藻在各个季节均有发现,玛氏骨条藻仅出现在春季.

Maltivariate Analysis of the Occurring Process of Skeletonema costatum Red Tide in the Changjiang Estuary

Using the SAS software, the data of environmental factors vs. red tide biomass were analysed during the process of Skeletonema costatum red tide which broke out in the Changjiang Estuary on June 9～15, 1990. The results of the multivariate statistical analysis show that the environmental factors are similar in their developing and maintenance periods and that the values of environmental factors begin to be restored to normal levels during the disappearing period.The results obtained from using the methods of correlation, variable cluster and main component analyses are basically identical. The important factors contributing to the occurrence of this red tide are atmospherical pressure, water temperature, salinity, pH, No3-, -N, Po43--P and Fe, among which,salinity, Fe and PO43--P are the leading factors which have triggered the occurrence of the red tide as determined by the step-by-step regression analysis. Meanwhile, the linear discrimination functions have been established for the red tide. The environmental factor data of another occurring process of Skeletonema costatum red tide in the Changjiang Estuary have been chosen for red tide discrimination with satisfactory results.

Large scale northward expansion of warm water species Skeletonema tropicum (Bacillariophyceae) in China seas

Skeletonema tropicum is regarded as a species with an affinity to warm waters and it has never been reported in seas where temperatures drop below 11℃ in winter. Previous studies in China reported that S. tropicum was restricted to subtropical and warm temperate seas （East and South China Seas）, but the species was recently found during August cruises of 2009 and 2010 in Jiaozhou Bay, Yellow Sea, located several hundred kilometers to the north. Here, winter water temperatures often drop below 5℃. Identification of S. tropicum was confirmed under light and scanning electronic microscopes and maximum cell abundance in Jiaozhou Bay was estimated as 1.73× 10^4 celI/L. This record of S. tropicum in Jiaozhou Bay represents a significant northward expansion in the geographic range of the species. Ship ballast water was identified as a possible carrier of S. tropicum from southern places along Chinese coastline, and in addition, thermal pollution from local power stations and seawater desalination plants may provide suitable conditions for species over-wintering.

Influences of external nutrient conditions on the transcript levels of a nitrate transporter gene in Skeletonema costatum

To verify the feasibility of high-affinity nitrate transporter gene （Nrt2） as an indicator of nitrogen status, changes in the transcript levels of transcripts associated with phosphate starvation and different nitrate concentrations were studied using real-time quantitative reverse-transcription PCR （QRT-PCR） technology in batch cultures of Skeletonema costatum. The results show that compared with P-replete condition, P starvation could reduce the Nrt2 transcript levels apparently. Nrt2 transcript levels had a significant negative linear correlation with nitrate concentrations below 40 pmol/L. The results of 48 h short-term incubation experiment under different nitrate concentrations confirmed this correlation, and the following regression equation is built： y = -3.305x ＋ 98.95, R2 = 0.988, where x represents nitrate concentrations （〈40 btmol/L） and y represents the Nrt2 transcript levels.

Influence of N, P, Fe Nutrients Availability on Nitrogen Metabolism-Relevant Genes Expression in Skeletonema marinoi

Nitrogen(N), Phosphorus(P), and Iron(Fe) are essential elements for cellular structure and metabolism. In addition to dissolved inorganic nitrogen(DIN), phytoplankton is able to utilize dissolved organic nitrogen(DON). There is general consensus that both bacteria and higher plants nitrogen metabolism is affected by phosphate availability; this was also found to be true in coccolithophorid. Iron affects the structure and function of ecosystems through its effects on nitrogen metabolism. However, it is unclear how these nutrients affect Skeletonema marinoi's nitrogen metabolism. Here, using RT-qPCR, we investigate effects of N, P, and Fe on S. marinoi's nitrogen metabolism and nitrate reductase activity. These results illuminate that in S. marinoi, various nutrients have direct regulation on these genes expression at the molecular level. The varying degree of responses for these genes expression with differing N sources may act to increase the efficiency of nutrient capture when nitrate is limited. Suitable gene expression occurs at a N/P ratio of 16, which represents the atomic N/P ratio of phytoplankton cells and N/P concentrations in ocean; thus, nitrogen metabolism gene expression should be regulated by the existing N/P ratios in the phytoplankton's internal and external environment. Fe concentration has a direct and significant effect on nitrogen metabolism by regulating gene expression and nitrate reductase activity. Gene expression profiles identified in S. marinoi provide a foundation for understanding molecular mechanisms behind diatom nitrogen metabolism with changing N, P, and Fe nutrients allowing a basic understanding of how diatom growth is affected by nutrient utilization.

Nutrient compositions of cultured Thalassiosira rotula and Skeletonema costatum from the Jiaozhou Bay in China

Carbon, nitrogen, phosphorus, silicon composition of cultured two different sized phytoplankton common species of Thalazsiosira rotula and Skeletonema costatum from the Jiaozhou Bay were measured. Carbon, nitrogen, phosphorus, silicon contents in cell were obvious higher in T. rotula than in S. costatum, but the percents of nitrogen, phosphorus, sihcon contents in cell dry mass in T. rotula were lower than those in S. costatum. The dry mass concentrations of nitrogen,phosphorus, silicon in S. costatum were much higher than those in T. rotula, particularly silicon, the former was 6. 4 times of the latter, showing that S. costatum could more assimilate these elements. Especially, S. costatum had competitive dominance for assimilating silicon, which is beneficial to its becoming a major dominant species in relative short silicon of the Jiaozhou Bay. There were some differences in numerical value of nutrient ratios both laboratory-cultured phytoplankton and different sized suspended particulates （ mainly phytoplankton） in the Jiaozhou Bay, which was caused by the changes of environment. High contents of carbon, nitrogon and relative low phosporus, sil- icon, high molar ratio of nitrogen to phosphorus （far higher than Redfield value） and low ratio of silicon to phosphorus and silicon to nitrogen （far lower than Redfield values） in the two diatoms and different sized suspended particulates were consistent with those in the seawater. Relative short silicon in the seawater and phytoplankton showed that silicon was possibly affectting phytoplankton growth in the Jiaozhou Bay.

Estimation of growth rate of Skeletonema costatum based on relative expression amount of PCNA gene

Partial sequence of Skeletonema costatum proliferating cell nuclear antigen （PCNA） gene was obtained by reverse transcriptase PCR（RT-PCR） and 3＇ rapid amplification of cDNA ends （3＇-RACE） techniques. Based on the obtained PCNA and cytochrome b gene（ Cyt b gene） sequences, a real-time fluorescent quantitative PCR （FQ-PCR） method was developed to detect the expression S. costatum PCNA gene, and this method was applied to study the relationship between the growth rate of S. costatum and the average expression amount of PCNA gene in a single cell. The expression amount of PCNA gene had large variation in cells collected at different culture phases, and the trend was well consistent with the growth rate, which suggested that the expression amount of PCNA gene correlated well with the cell division, and the PCNA could be a promising indicator for the S. costatum cell proliferation. Furthermore, using the PCNA gene as the objective gene and the Cyt b gene as the house-keeping gene, a new method for estimating the in situ growth rate of S. costatum was established by analysis of the relative expression quantity （REQ） of the PCNA gene.

Competition Among Dinoflagellate Alexandrium Tamarense, Raphidophyte Heterosigma Carterae and Diatom Skeletonema Costatum under Combinations of Two Temperatures and Five Salinities

Competition among HAB (Harmful Algal Bloom) species Dinoflagellate Alexandrium tamarense , Raphidophyte Heterosigma carterae , and Diatom Skeletonema costatum was studied in the laboratory. Experiments with these three major HAB species under combinations of different salinities (10, 18, 25, 30, 35) and temperatures (19℃, 25℃) were carried out. The results showed that S. costatum successfully competed with the other two species at salinities of 18, 25, 30, and 35 at temperatures of 19℃ and 25℃. However, H. carterae showed its advantage at low salinity of 10 and became the single dominant species at salinity 10 and 25℃. A. tamarense could not compete successfully with the other two species especially at low salinities. However, it could remain at low density in the presence of higher densities of other algae.

Study on the Effect of Monochromatic Light on the Growth of the Red Tide Diatom <i>Skeletonema Costatum</i>

Effects of light intensity and quality of three kinds of LED monochromatic lights (blue, green, and red) on the growth of Skeletonema costatum are investigated in batch culture conditions. Seven light intensities (20, 30, 40, 45, 50, 60 and 80 μmol·m-2·s-1) are used to evaluate the specific growth rate, spectrum absorption coefficient and saturated light intensity of LED monochromatic light. Results show that the growth rates of Skeletonema costatum increase with the enhanced light intensity;however, the light level beyond the saturation light intensity inhibited the growth of Skeletonema costatum. Compared with red and green light, the growth rate of Skeletonema costatum under blue light is higher within saturated light intensity, and saturated light intensity of LED monochromatic light is lower under blue light and higher under green light. It is concluded that under different monochromatic light, the saturated light intensity decreases and the growth rate increases with the increasing of spectrum absorption coefficient.