日照贝类养殖区海水微生物群落结构特征及其影响因素

微生物群落结构和组成差异是反映海洋生态系统环境健康的有效指标,对生态环境监测和管理具有重要意义,同时可以为水产养殖活动提供启示。本文针对日照某贝类养殖区及非养殖区水体环境的微生物群落结构进行了测序和分析,并探讨了环境因子对微生物群落结构和多样性的影响。结果显示,变形菌门（Proteobacteria）是养殖区海水的优势细菌类群,其次是蓝细菌门（Cyanobacteria）和放线菌门（Actinobacteria）。养殖区表层海水中蓝细菌门的相对丰度（P=0.003）以及8 m深度水层中变形菌门的相对丰度（P=0.027）显著高于非养殖区。微生物多样性分析表明,4月至11月养殖区表层及8 m水层的微生物Shannon指数范围分别为5.59～6.92和5.33～7.08,与非养殖区无明显差异,但养殖区的微生物丰富度指数（表层Chao1=1 088.86,8 m深度水层Chao1=1 158.77）略低于非养殖区（表层Chao1=1 108.63,8 m深度水层Chao1=1 210.30）。典型相关性分析（CCA）和Spearman相关性分析结果表明,温度（P=0.003）、盐度（P=0....     

海南红树林根系土壤中可培养细菌的多样性分析

为研究海南红树林根系土壤细菌多样性,充分发掘中国红树林特有微生物资源,作者于2012年12月份从10份采集于海南北港岛红树林根系淤泥样品中分离可培养海洋细菌,并基于16S rRNA基因序列分析的方法对样品中的细菌多样性进行研究。共分离得到122株海洋细菌,经并菌后选取其中的70株代表性菌株测序,结果发现它们分为4个类群:变形菌门(主要为γ-变形菌纲)(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),分别占66%,19%,11%和4%。共代表30个属47个种,其中有9个潜在新种和2个潜在新属。本研究结果表明,北港岛红树林中存在着丰富的细菌多样性,很多类群可能是红树林特有细菌物种。     

山东省渤海海洋保护区典型海域表层海水微生物群落多样性分析

海洋微生物在生态系统循环中发挥重要作用。为系统了解山东省渤海海洋保护区表层海水微生物群落多样性,作者利用Illumina高通量测序及生物信息学分析技术研究了12个水体样本的菌群构成和多样性特征。实验结果表明,山东渤海海洋保护区典型海域表层海水微生物群落丰富,12个水体样品OTU分属于25门、56纲、103目、163科、227属;门、纲、目、科、属分类等级上的优势类群分别为变形菌门(Proteobacteria)(57.95%)、α-变形菌纲(Alphaproteobacteria)(21.88%)、黄杆菌目(Flavobacteriales)(4.31%)、红细菌科(Rhodobacteraceae)(10.68%)、Candidatus portiera(7.94%);保护区内表层海水微生物具有其独特的菌群特征和多样性。本研究结果为进一步认识海洋微生物多样性与海洋生态环境保护的关系奠定了基础。     

基于高通量测序的象山港水域细菌多样性的研究

象山港位于浙江省宁波市东南部沿海,属于半封闭式海湾,是以海水养殖区和海洋牧场示范区为主的重要多功能水域,近年来生态环境问题日益突出。为研究象山港海域在早春低水温环境中细菌的多样性,通过高通量测序方法对3月份的象山港水域进行检测。沿象山港南部海湾随机选取6个取样点(G04,G06,G08,G09,G011,G012),共检出836属和2 166种细菌,隶属33个门、91纲、186目和352科。其中优势细菌类群主要包括变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和蓝细菌门(Cyanobacteria)。变形菌门在G04中占比最高(91.45%),拟杆菌门在G09中占比最高(18.05%);而放线菌门在G12中占比最高(7.95%)。在经度较高且离海岸较远的水域,细菌丰富度也越高;同时随着取样位点纬度的增加,γ-变形菌丰度逐步增高,其中在G04中最高(72.76%),在G12中最低(29.11%)。各取样点的细菌分离培养结果也表明弧菌属丰度较高,且主要集中在G04中。利用高通量测序方法揭示了象山港早春全水域的细菌结构组成框架图,可为象山港的水产养殖和可持续发展提供参考依据。     

热带东印度洋春季浮游植物群落结构空间特征分析

基于2013年3月至5月采集的热带东印度洋海域(10.0°S～4.0°N, 83.0°～97.5°E)浮游植物水样样品，分析了其种类组成、优势类群、细胞丰度等群落特征参数，综合比较了水平和垂向上浮游植物种类及丰度的差异性，初步探讨了其成因。结果表明:2013年春季热带东印度洋微型浮游植物共鉴定出306种，主要由硅藻、甲藻、金藻、蓝藻、裸藻和隐藻六大门类组成，其优势类群主要以粒径较小的隐藻、微型甲藻、菱形藻、环沟藻等为主。水平分布上，各水层浮游植物细胞丰度分布趋势相似，但斑块特征明显，其高值区位于88°E断面赤道以南次表层水域(30 m、75 m)，局部区域细胞丰度值可达104 cells/L以上；与其毗邻的东南部、东部水域为低值区，并向赤道延伸。垂直剖面上，硅藻和甲藻广泛分布在各取样深度，但分布特征有明显的空间差异和规律，0 m、30 m大部分站位硅藻种类比例在0.2～0.3甚至更低，甲藻在0.7～0.8，随着水层加深(75 m、100 m、150 m、300 m)，硅藻种类占比上升到0.5～0.6，甲藻降低到0.4～0.5，无论硅藻还是甲藻种类数75 m层最丰富。30 m和75 m水层细胞丰度明显高于其他水层。甲藻是热带东印度洋微型浮游植物种类和细胞丰度的重要贡献者，低生物量海域表现的更为明显，贡献率大于80%。该研究将极大丰富东印度洋浮游植物群落空间特征基础信息较匮乏的现状，为量化、评估该海域的生物资源提供数据支撑。     

泰国海域儒艮发声分类及特征

为研究近海扇贝养殖水域水质、细菌种群分布规律以及微生物群落多样性对环境因子的响应,2019年5月至10月采集了青岛市崂山湾青山村近海栉孔扇贝养殖水域不同站位的海水样本,通过高通量测序方法分析了不同月份海水样本微生物群落结构和多样性的差异,探讨了环境因子与微生物群落结构的相关性。结果表明:①检测到变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、蓝细菌门(Cyanobacteria)、放线菌门(Actinobacteria)和厚壁菌门 (Firmicutes)等 52 个 细 菌 门,115 个 纲,其 中 变 形 菌 门 为 优 势 菌 门,主 要 包 括 γ-Proteobacteria 和α-Proteobacteria纲;②2019年9月,养殖区水样中细菌多样性升高,弧菌多样性下降,且细菌细胞数量达到最高点,说明该 养 殖 区 存 在 爆 发 弧 菌 病 的 可 能 性;③RDA 冗 余 分 析 表 明,浊 度 (P =0.027)对 养 殖 密 区 细 菌 OUT(OperationalTaxonomicUnits)水平的物种组成和群落结构具有显著的影响,硝酸盐(P=0.017)、浊度(P=0.033)对养殖疏区 OTU 水平的物种组成和群落结构具有显著的影响;④不同区域细菌门水平环境因子相关性 Heatmap图分析表明,养殖活动会影响近海细菌群落结构与环境因子的作用程度,从而增加病害发生的可能性。本研究可为贝类疾病的防治及近海海域生态系统的健康发展提供一定的理论基础。     

浙江镇海潮间带沉积物中可培养微生物初步研究

针对浙江镇海潮间带沉积物样 品,采用纯培养法分离培养海洋微生物,并基于16S rRNA基因序列,开展系统发育学研究,分析沉积物细菌群落结构及多样性。分离获得细菌39株,16S rRNA基因序列分析表明,这些菌株分别属于厚壁菌门Firmicutes(51. 3%)、变形杆菌门Proteobacteria(30.8%)、拟杆菌门Bacteroidetes(15. 4%)和放线菌门Actinobacteria(2. 6%)4个类群。厚壁菌门和变形杆菌门的菌株主要归属于芽孢杆菌纲Bacilli和a-变形杆菌纲Alphaproteobacteria,它们可能在近海海洋生态系统的元素地球化学循环中发挥了重要作用。此外,部分菌株16S rRNA基因序列与已报道物种的相似性较低,可能代表了新的分类单元。研究丰富了浙江近海微生物种质资源,并拓展了对浙江近海可培养细菌多样性的认识,为今后开发利用海洋微生物资源积累了资料。     

南海北部水层间及沉积表层细菌群落的比较分析

细菌群落在海洋生物地球化学过程中起着至关重要的作用。分析判别地理距离和环境条件等因素对细菌群落的影响,有助于理解环境变化如何驱动微生物多样性和功能的机制。通过细菌16SrDNA高通量测序的方法,对南海北部水层及沉积表层细菌群落多样性及空间分布进行比较分析,并检验环境因素、地理限制及海底深度等因素对微生物群落的影响。结果表明,约95%的细菌16SrDNA序列归属于γ-变形菌纲、厚壁菌门、蓝细菌门、α-变形菌纲等优势类群。沉积样本中的优势细菌类群主要与粒度、氮(N)等环境参数相关,而水体样本的优势细菌类群与取样深度密切相关。相关性检验结果显示,沉积样本的α-多样性指数与C有着显著的相关关系,而水体样本的α-多样性指数和β-多样性矩阵则显示与取样深度的显著相关;此外,地理距离和海底深度对β-多样性的影响并不显著,这可能与取样空间尺度有关。     

北极黄河站邻近海域浮游细菌多样性初步研究

通过荧光染色、稀释培养、生理生化和16SrRNA基因鉴定等方法,研究了北极黄河站附近海域海水中浮游细菌的丰度、可培养浮游细菌形态学特征及多样性;采用高通量测序技术研究了黄河站附近采样站点(BJ2)的不可培养浮游细菌群落结构。可培养研究结果表明:(1)9个站点(BJ1-BJ9)水样平均含菌数为2.88×108个/L;(2)21株可培养浮游细菌中,仅AB08、AB09和AB17三株细菌为革兰氏阳性菌;(3)细菌主要包括放线菌纲(Actinobacteria),黄杆菌纲(Flavobacteriia)和γ-变形菌纲(Gammaproteobacteria),其中假单胞菌属(Pseudomonas)为优势菌(占71%)。高通量测序结果显示:在得到的1 467个不可培养浮游细菌OTU中,变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)为优势菌群,所占比例分别为49.71%和41.61%,还有少量的浮霉菌门(Planctomycetes)和疣微菌门(Verrucomicrobia);其中变形菌门中以α-变形菌纲(α-Proteobacteria)为主要类群。     

九龙江河口区微生物多样性及群落结构的时空分布

河口区由于其独特的地理环境和理化条件,拥有丰富的微生物资源,在生物地球化学循环中起着重要作用.然而,由于人类活动带来的河口区环境因素改变,引起的微生物群落结构的时空变化目前还知之甚少.本研究选取九龙江河口区7个近年来遭受较为严重人类活动干扰的采样点,分别在丰水期和枯水期采集表层水体,采用流动注射法测定了水体的三氮、电导率、pH值和溶解性磷酸盐等环境参数,采用海水和淡水培养基,基于纯培养技术分析了可培养细菌的总数和分布特征,并通过构建16S rRNA基因克隆文库的方法研究细菌的多样性和群落结构变化.研究表明,变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)是各克隆文库中最优势的类群.在河口下游海水区,变形菌门微生物与放线菌门微生物的比例约为2∶1~3∶1,而在河口上游淡水区,变形菌门和放线菌门的比例约为1∶1.在河口下游海水区,α变形菌纲(Alphaproteobacteria)为变形菌门中的优势类群,而在河口上游淡水区,β变形菌纲(Betaproteobacteria)为优势类群.厚壁菌门(Firmicutes)是克隆文库中丰度占第四的类群,说明水体可能遭受畜牧养殖粪便污染.本研究表明,九龙江河口区微生物群落结构受水体盐度、温度、水文情况等时空因子及人类活动造成的营养物浓度上升、动物粪便污染等共同影响,呈现出独特的时空分布特点.     

西南印度洋深海热液硫化物区沉积物微生物群落结构和固氮基因多样性

A sediment sample was collected from a deep-sea hydrothermal vent field located at a depth of 2 951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community using polymerase chain reaction（PCR） amplification of the 16 S rRNA and nifH genes. Within the Archaea, the dominant clones were from marine benthic group E（MBGE） and marine group I（MGI） belonging to the phyla Euryarchaeota and Thaumarchaeota, respectively. More than half of the bacterial clones belonged to the Proteobacteria, and most fell within the Gammaproteobacteria. No epsilonproteobacterial sequence was observed. Additional phyla were detected including the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3 and TM6, confirming their existence in hydrothermal vent environments. The detection of nifH gene suggests that biological nitrogen fixation may occur in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis indicated that only Clusters I and III NifH were present. This is consistent with the phylogenetic analysis of the microbial 16 S rRNA genes, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.     

福建漳浦凡纳滨对虾海水养殖中后期水体细菌群落多样性分析

为分析凡纳滨对虾（Litopenaeus vannamei）海水养殖过程中水体细菌群落多样性变化,本研究采用Illumina测序技术对福建漳浦县凡纳滨对虾海水养殖中后期水体样品进行16S rRNA基因高通量测序,同时进行水质参数测定。结果表明,养殖过程中,氨氮（NH₄+-N）、化学需氧量（COD）值波动较大,其他水质参数值波动较小。检测到的细菌种群归属于19个门、35个纲、80个目、135个科、254个属,说明该养殖水体中细菌群落具有高度的多样性。变形菌门和拟杆菌门是养殖期间主要优势类群,不同养殖时期细菌组成存在一定的波动,但没有明显的变化规律。蓝细菌在第67天、115天表现出高丰度,且其丰度在养殖期间变化较大（比例变化为0.7%~63.9%）。此外,益生菌和潜在致病菌丰度在养殖期间很低。冗余分析（Redundancy analysis,RDA）表明,对细菌群落影响最大的水质因子是化学需氧量与硝酸盐浓度比值（COD/NO₃-）和COD;降低溶氧可以增加细菌丰富度。总之,本研究初步揭示了对虾养殖中后期水体微生物的演替规律,发现水质因子对养殖水体微生物组成具有显著影响,研究结果为凡纳滨对虾健康养殖提供一定的参考意义。     

Influence of the Po River runoff on the bacterioplankton community along trophic and salinity gradients in the Northern Adriatic Sea

The Po River runoff strongly affects the oceanographic and ecological characteristics of the Northern Adriatic Sea. Catalysed reported deposition ‐ fluorescence in situ hybridization (CARD‐FISH) analysis was employed to assess how the composition of the coastal bacterioplankton community is influenced by the river runoff in two different seasons (spring and autumn). Samples were collected from the water column along a coastal–offshore transect in the Northern Adriatic Sea at different depths. Four clone libraries were then constructed from coastal (0 m) and offshore waters (?65 m). Higher abundances of bacteria were recorded in coastal waters as compared with the offshore samples. This result was mainly due to the trophic state of the water column, and it was related to salinity. Particularly, Actinobacteria and Gammaproteobacteria were affected by the riverine inputs, whereas Bacteroidetes and Alphaproteobacteria showed only minor responses. This was particularly clear in the autumn sample, in which a clear difference between the coastal and the offshore samples was found due to a strong influence of the less saline river water with high nutrient concentrations. Analysis of 205 partial length 16S rRNA gene sequences indicated a high diversity with the dominance of Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria mainly affiliated to coastal and marine bacterioplankton clades. Actinobacteria were also detected and, together with Gammaproteobacteria, strongly follow the freshwater intrusion. Overall, our results indicate that the seasonal difference in the Po River discharge greatly affects the bacterioplankton community. In spring we observed a smooth transition from coastal to open‐sea conditions owing to the more superficial freshwater plume. In autumn the deeper freshwater intrusion in the coastal site and thermal stratification offshore resulted in a marked difference between the coastal and offshore microbial communities.     

东印度洋现生浮游有孔虫

A brief morphometric study of the recent planktonic foraminifera in the eastern Indian Ocean was provided with the taxonomic key to species,synonyms,SEM microphotographs of shells and chamber arrays.By recent classification,currently 20 species representing 13 genera and 6 families(Canderinidae,Heterohelicoidae,Hedbergellidae,Higerigerinoidae,Globigerinoidae,and Globorotaloidae)identified from the planktonic material of the eastern Indian Ocean up to a depth of 200 m.Their distribution in water(0–200 m)also reports on the new range of expansion in the eastern Indian Ocean,with Dentigloborotalia anfracta,Hastigerina pelagica,Streptochilus globigerus,Globigerinella calida,Globigerinella adamsi,Orcadia reidelii,Tenuitella parkerae,Tenuitella compressa,reported for the first time in this study area.In general,only around 50 planktonic species are valid worldwide,more specifically the species,e.g.,H.pelagica,G.calida,G.adamsi,S.globigerus,O.riedeli,T.parkerae,T.compressa,which occur in the eastern Indian Ocean to fill the the paucity of the recent regional taxonomic literature and the problematic identification from the eastern Indian Ocean.This work aims to bridge this gap and help scientists,managers,educators and students to identify plankton foraminifers by using species notes and images.     

东印度洋冬季印尼贯穿流影响区浮游植物群落结构特征

东印度洋受到印尼贯穿流(Indonesian Throughflow,ITF)等多个大洋流系影响,是太平洋水在低纬度进入印度洋的重要通道,具有显著的生态效应,是金枪鱼的重要渔场之一。为了解上述海域浮游植物群落组成、时空特征及其影响因素,基于2020年1～2月在该海域采集到的浮游植物样品及同步的环境数据,对其种类组成、生态类型、细胞丰度、优势类群等群落组成及其与环境因子关系进行了研究,并与赤道东印度洋浮游植物群落进行了比较。结果表明:研究海域共鉴定浮游植物4门57属268种(含变种、变型),其中,硅藻38属129种,甲藻17属136种,金藻1属2种,蓝藻1属1种;生态类型以热带大洋性种类、大洋暖水种和热带近岸种为主,浮游植物区系属于热带-亚热带生物区系。细胞丰度介于1.28×10³～7.84×10⁴ cells/m³之间,平均值为1.55×10⁴ cells/m³。优势种中硅藻占绝大多数,分别为短叉角毛藻(Chaetoceros messanensis)、伏氏海毛藻(Thalas...     

Microbial Diversity of Deep-sea Sediments from Three Newly Discovered Hydrothermal Vent Fields in the Central Indian Ridge

Since the discovery of hydrothermal vents in the late 1970s, deep-sea hydrothermal vent fields have attracted great attention as biological hotspots. However, compared with other ocean ridges, the structure and function of microbial communities inhabiting vent fields in the Central Indian ridge (CIR) remain understudied. Here, we provide for the first time 16S rRNA gene-based comparative metagenomic analysis of the sediment-associated microbial communities from three newly discovered vent fields in the CIR. Sediment samples collected in the Invent B, Invent E and Onnuri vent fields varied in geochemical properties, elemental concentrations and associated microbial communities. Proteobacteria (Gammaproteobacteria) was the dominant phylum in Invent B and Onnuri vent fields. In contrast, Invent E mainly consisted of Chloroflexi and Euryarchaeota. Predicted functional profiling revealed that the microbial communities in the three vents are dominated by chemoheterotrophic functions. In addition, microbial communities capable of respiration of sulfur compounds, nitrification, nitrite oxidation, methylotrophy, and methanotropy were found to be the main chemolithoautotrophs. Compared to other vent fields, Invent E showed a predominance of archaeal methanogens suggesting it exhibits slightly different geochemistry. Multivariate analysis indicated that the biogeochemical and trace metal differences are reflected in the sediment microbial compositions of the three vent fields. This study expands our current understanding of the microbial community structure and potential ecological functions of the newly discovered hydrothermal vent fields in the CIR.

     

Phytoplankton composition and biomass across the southern Indian Ocean

Phytoplankton composition and biomass was investigated across the southern Indian Ocean. Phytoplankton composition was determined from pigment analysis with subsequent calculations of group contributions to total chlorophyll a (Chl a) using CHEMTAX and, in addition, by examination in the microscope. The different plankton communities detected reflected the different water masses along a transect from Cape Town, South Africa, to Broome, Australia. The first station was influenced by the Agulhas Current with a very deep mixed surface layer. Based on pigment analysis this station was dominated by haptophytes, pelagophytes, cyanobacteria, and prasinophytes. Sub-Antarctic waters of the Southern Ocean were encountered at the next station, where new nutrients were intruded to the surface layer and the total Chl a concentration reached high concentrations of 1.7 ??g Chl a L⁻¹ with increased proportions of diatoms and dinoflagellates. The third station was also influenced by Southern Ocean waters, but located in a transition area on the boundary to subtropical water. Prochlorophytes appeared in the samples and Chl a was low, i.e., 0.3 ??g L⁻¹ in the surface with prevalence of haptophytes, pelagophytes, and cyanobacteria. The next two stations were located in the subtropical gyre with little mixing and general oligotrophic conditions where prochlorophytes, haptophytes and pelagophytes dominated. The last two stations were located in tropical waters influenced by down-welling of the Leeuwin Current and particularly prochlorophytes dominated at these two stations, but also pelagophytes, haptophytes and cyanobacteria were abundant. Haptophytes Type 6 (sensuZapata et al., 2004), most likely Emiliania huxleyi, and pelagophytes were the dominating eucaryotes in the southern Indian Ocean. Prochlorophytes dominated in the subtrophic and oligotrophic eastern Indian Ocean where Chl a was low, i.e., 0.043-0.086 ??g total Chl a L⁻¹ in the surface, and up to 0.4 ??g Chl a L⁻¹ at deep Chl a maximum. From the pigment analyses it was found that the dinoflagellates of unknown trophy enumerated in the microscope at the oligotrophic stations were possibly heterotrophic or mixotrophic. Presence of zeaxanthin containing heterotrophic bacteria may have increased the abundance of cyanobacteria determined by CHEMTAX.     

厚壳贻贝(Mytilus coruscus)养殖海域与天然生长海域的微生物群落比较研究

为研究厚壳贻贝(Mytilus coruscus)生长海域微生物群落的结构与多样性特征,初步探究海域微生物群落及共生菌对贻贝生长的影响,分别采集舟山市东极镇庙子湖岛和嵊泗县枸杞岛厚壳贻贝养殖区和野生生长区海水样品,利用16S rRNA扩增子测序技术,比较分析不同海域微生物群落的组成及丰度特征以及微生物群落的差异;同时对厚壳贻贝体内的微生物进行了初步的分离鉴定。研究结果显示,嵊泗厚壳贻贝野生生长海域10 m水深的微生物丰富度显著大于5 m水深,东极厚壳贻贝养殖区海域10 m水深微生物丰富度也而明显大于5 m水深。东极和嵊泗厚壳贻贝的生长海域形成了较为稳定微生物群落结构,嵊泗海域主要优势菌群为十八杆菌属Octadecabacter、假交替单胞菌属Pseudoalteromonas、厌氧氨氧化菌属Candidatus_Portiera、沉积物陆丹氏菌属Loktanella和弧菌Vibrio;东极海域主要优势菌群为厌氧氨氧化菌属Candidatus_Portiera、海洋氨氧化古菌属Nitrosopumilus、蓝细菌聚球藻属Synechococcus和弧菌属Vibrio。PCA分析结果表明东极和嵊泗两个海域的厚壳贻贝野生生长区的微生物群落相似度明显低于养殖区的微生物群落相似度,且这种差异在嵊泗海域尤为明显。同时在贻贝体内分离出了假交替单胞菌、黏着杆菌以及弧菌,而这三种菌在海水样品中也大量存在,由此可初步判定这三类细菌能够随水流进入贻贝体内,并可能对贻贝的附着、生长以及防病等生理功能产生一定的影响。