A theoretical individual-based model of Brown Ring Disease in Manila clams,Venerupis philippinarum

An individual-based mathematical model was developed to investigate the biological and environmental interactions that influence the prevalence and intensity of Brown Ring Disease (BRD), a disease, caused by the bacterial pathogen, Vibrio tapetis, in the Manila clam (Venerupis (= Tapes, = Ruditapes) philippinarum). V. tapetis acts as an external microparasite, adhering at the surface of the mantle edge and its secretion, the periostracal lamina, causing the symptomatic brown deposit. Brown Ring Disease is atypical in that it leaves a shell scar that provides a unique tool for diagnosis of either live or dead clams. The model was formulated using laboratory and field measurements of BRD development in Manila clams, physiological responses of the clam to the pathogen, and the physiology of V. tapetis, as well as theoretical understanding of bacterial disease progression in marine shellfish. The simulation results obtained for an individual Manila clam were expanded to cohorts and populations using a probability distribution that prescribed a range of variability for parameters in a three dimensional framework; assimilation rate, clam hemocyte activity rate (the number of bacteria ingested per hemocyte per day), and clam calcification rate (a measure of the ability to recover by covering over the symptomatic brown ring deposit), which sensitivity studies indicated to be processes important in determining BRD prevalence and intensity. This approach allows concurrent simulation of individuals with a variety of different physiological capabilities (phenotypes) and hence by implication differing genotypic composition. Different combinations of the three variables provide robust estimates for the fate of individuals with particular characteristics in a population that consists of mixtures of all possible combinations.The BRD model was implemented using environmental observations from sites in Brittany, France, where Manila clams routinely exhibit BRD signs. The simulated annual cycle of BRD prevalence and intensity agrees with observed disease cycles in cultured clam populations from this region, with maximum disease prevalence and intensity occurring from December to April. Sensitivity analyses of modeled physiological processes showed that the level of hemocyte activity is the primary intrinsic determinant of recovery of infected clams. Simulations designed to investigate environmental effects on BRD suggested that the outcome of the host–parasite interaction is dependent on food supply (high values being favorable for the host) and temperature. Results of simulations illustrate the complex interaction of temperature effects on propagation and viability of the bacterium, on the phagocytic activity of the hemocytes, and on other physiological processes of the host clam. Simulations using 1 °C and 2 °C increases in temperature generally favored disease development, indicating that climate warming might favor the spread of BRD.     

Hemocytes of Mytilus edulis affected by Prudhoe Bay crude oil emulsion

Hemocytes and tissues of Mytilus edulis were examined after 4–5 or 8–9 week's exposure to 390 μg/liter or 740 μg/liter Prudhoe Bay crude oil emulsion, during the animals' most metabolically active season. A reduction in hemocytes occurred in animals exposed to 740 μg/liter after 4–5 weeks. After 8–9 weeks, hemocyte counts of both test groups increased, due to higher densities of granulocytes, yet the phagocytic response was reduced. Agranulocyte densities were reduced in animals exposed to 390 μg/liter, due to lower counts of 2–3 μm agranulocytes. Further, adipogranular cell percentages in test animals were reduced. The results from oil-exposed mussels suggest a stress condition and have implications for monitoring.     

日本蟳血细胞及血液生化指标的研究

以光镜和电镜观察了日本蝠（Charybdisjaponica）血细胞的形态结构,分析了各类血细胞的大小、数量比例及功能,并测定了不同发育时期及饥饿状态的日本蝽血清主要生化指标。结果表明：日本蝠血细胞可分为4种类型,从小到大依次为：无颗粒细胞、小颗粒细胞、中间型细胞和大颗粒细胞;其核质比分别为：53．23％、33．64％...     

Total hydrocarbons,polycyclic aromatic hydrocarbons and synthetic organic compounds in the Hard shell clam, Mercenaria mercenaria, purchased at commercial seafood stores

An analytical method for the determination of polycyclic aromatic h hydrocarbons (PAHs) and substituted benzotriazoles in clams was developed using a combination of silica gel and Sephadex chromatography to isolate these compounds from large concentrations of polyolefinic hydrocarbons. The concentrations of total hydrocarbons, PAHs and substituted benzotriazoles were measured in hard shell clams (Mercenaria mercenaria) purchased from Rhode Island seafood stores. The levels of total hydrocarbons, PAHs and substituted benzotriazoles in these clams were generally higher than the concentrations found in clams collected from a lower Narragansett Bay control location. The significance of these findings on the health of consumers is unknown at this time because human health standards or alert levels for these compounds in seafoods have not been established. However, this study illustrates that contaminants entering Narragansett Bay from a variety of sources may eventually make their way to commercial outlets.     

Copper bioavailability to marine bivalves and shrimp: Relationship to cupric ion activity

Studies were performed to determine the effects of dissolved substances present in natural seawater and sediment on the bioavailability of added Cu⁺². Whole clams Macoma inquinata and shrimp, Pandalus danae, were exposed to four concentrations of Cu in a flow-through seawater system. Bioaccumulation of Cu was reduced in shrimp, Pandalus danae, clams, M. inquinata, and excised clam gills, Protothaca staminea, exposed to an aged, compared with an unaged, Cu-seawater solution. This is thought to be due to slow complexation of the Cu by dissolved substances present in natural seawater. In a static system, with added sediment, more than 50% of the added Cu⁺² became bound to the organic fraction of the sediment and was unavailable to suspension feeding clams, Protothaca staminea. In contrast, deposit feeding clams, Macoma inquinata, placed in the sediment approximately doubled in Cu body burden within two months.Complexed Cu appears to be less bioavailable than ionic Cu and hence measurements of ionic and weakly complexed Cu by differential pulse anodic stripping voltametry provide a better prediction of bioavailable Cu than conventional measurements of total Cu-seawater concentrations.     

Assessment of immune parameters of manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> in different physiological conditions using flow cytometry

Cellular and humoral immune parameters are often used as biomarkers to trace environmental and physiological stresses in marine bivalves. In this study, we compared various immune parameters of Manila clams (Ruditapes philippinarum) under normal conditions and under a high level of desiccation, using flow cytometry. The immune parameters analyzed included, total hemocyte count, hemocyte mortality, hemocyte DNA damage, reactive oxygen species (ROS) production, and phagocytosis activity. Total hemocyte count, hemocyte DNA damage, and hemocyte mortality were significantly elevated among clams under high desiccation stress, while phagocytosis activity and spontaneous ROS production were significantly lower compared to those parameters of the control clams (p<0.05). These data suggest that the immune parameters analyzed in this study well reflect the physiological status of clams.     

杂色鲍血细胞分类、结构和免疫功能的流式细胞术分析

应用流式细胞术对杂色鲍(Haliotis diversicolor Reeve)血细胞的分类、结构和免疫功能进行分析。根据细胞前向角散射光(FSC)和侧向角散射光(SSC)强度的不同,可将血细胞分为三个亚群:透明细胞、小颗粒细胞和大颗粒细胞,组成比例分别为32.71%、58.17%和8.55%。血细胞的平均总凋亡和死亡率为3.76%。血细胞对荧光微球的总吞噬率为63.67%,其中吞噬1个、2个、3个及以上荧光微球的血细胞分别占22.31%、16.39%、24.96%。线粒体数量、溶酶体数量、非特异性酯酶活性和非诱导性活性氧(ROS)含量均在大颗粒细胞中最高,透明细胞最低。结果表明,杂色鲍三类血细胞在结构和功能上均存在差异,两类颗粒细胞可能在鲍类免疫过程中发挥着更为重要的作用。     

Histopathologic biomarker response of clam, <Emphasis Type="Italic">Ruditapes decussates</Emphasis>, to organophosphorous pesticides reldan and roundup: A laboratory study

Clam Ruditapes decussates was used as a biomonitoring organism to measure the potential impact of pollution. The present study investigated histological alterations of R. decussates following a long-term exposure to a sublethal-level of pesticides Reldan and Roundup. The clams were taken to the laboratory and kept for days to adjust to a continuous closed flow system with quality water control. After the adjustment period, clams were exposed either to sublethal concentrations of Reldan (0.6 μl L^?1) or Roundup (1.1 μl L^?1) for 90 days. Clams were taken to determine the histological damage at 30, 60 and 90 days. Most histological alterations were highly localized in the glandular cells of the gills, mantle tissues and digestive gland; secretions of glandular cells were found to be increased by increasing the time of exposure. Histopathological results showed the activation of resistance mechanisms that allowed the clams to survive under sublethal stresses. Thus, histological changes on localized tissues were sensitive and they have positive correlation to the time of exposure to the pesticides, suggesting that they may serve as biomarkers for Reldan and Roundup exposures.     

LPS胁迫下五个群体菲律宾蛤仔TYR基因在鳃和肝胰腺中表达特性研究

为了解TYR基因与蛤仔免疫的关系,本实验利用荧光定量PCR技术研究了菲律宾蛤仔五个群体(白蛤、白斑马蛤、斑马蛤、养殖和野生群体)的鳃组织和肝胰腺组织在LPS胁迫下TYR基因在不同时间(0 h、3 h、12 h、24 h、48 h)的表达特性。结果表明,在LPS注射后鳃组织中TYR6基因表达水平在白蛤和白斑马蛤3 h、6 h、12 h,野生蛤仔3 h,斑马蛤3 h、6 h,养殖群体6 h、12 h显著上调(P<0.05),3h野生蛤仔和斑马蛤达到峰值, 6 h白蛤、白斑马蛤、养殖群体达到峰值(P<0.05);在肝胰腺中,养殖群体和白斑马蛤6 h,白蛤6 h、24 h,野生群体24 h,斑马蛤3 h显著上调, 3 h野生蛤仔和斑马蛤达到峰值,6h养殖群体、白蛤、白斑马达到峰值(P<0.05);鳃组织中TYR10基因表达水平在白蛤、野生群体和白斑马蛤3 h,养殖群体3 h、6 h,斑马蛤3 h、12 h显著上调(P<0.05),肝胰腺组织中TYR10基因表达水平在白蛤3 h,野生群体3 h、6 h、12 h,斑马蛤、养殖群体6 h显著上调(P<0.05),推测T...     

Physiological and biochemical responses of <Emphasis Type="Italic">Prorocentrum minimum</Emphasis> to high light stress

Prorocentrum minimum is a common bloomforming photosynthetic dinoflagellate found along the southern coast of Korea. To investigate the adaptive responses of P. minimum to high light stress, we measured growth rate, and generation of reactive oxidative species (ROS), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in cultures exposed to normal (NL) and high light levels (HL). The results showed that HL (800 μmol m^?2 s^?1) inhibited growth of P. minimum, with maximal inhibition after 7–9 days. HL also increased the amount of ROS and MDA, suggesting that HL stress leads to oxidative damage and lipid peroxidation in this species. Under HL, we first detected superoxide on day 4 and H₂O₂ on day 5. We also detected SOD activity on day 5 and CAT activity on day 6. The level of lipid peroxidation, an indicator of cell death, was high on day 8. Addition of diphenyleneiodonium (DPI), an NAD(P)H inhibitor, decreased the levels of superoxide generation and lipid peroxidation. Our results indicate that the production of ROS which results from HL stress in P. minimum also induces antioxidative enzymes that counteract oxidative damage and allow P. minimum to survive.     

The Effect of an Infaunal Suspension Feeding Bivalve Mercenaria mercenaria (L.) on Benthic Recruitment*

Abstract. Animal-animal interactions are recognized as critical in benthic invertebrate recruitment. The hypothesis tested was that benthic recruitment is more successful in low densities of infaunal suspension feeders than in high densities. Densities of young hard clams Mercenaria mercenaria (L.) ranging from 82.5 to 330m^-2 were placed in defaunated boxes of sand. The mean number of species, mean number of individuals, mean wet weight biomass, mean species richness and mean dominance index per sample were calculated per density of hard clams from May to October. The experiment did not support the hypothesis. It was concluded that feeding behaviour of dense populations of M. mercenaria did not preclude successful recruitment of other benthic species.     

长牡蛎新型模式识别受体CgLRRC69的克隆和功能鉴定

在双壳类软体动物中, 血淋巴细胞介导的吞噬作用是清除入侵微生物的主要方式。本文在长牡蛎中鉴定了一个包含富含亮氨酸重复序列(Leucine-rich repeat, LRR)结构域的新型基因, 命名为CgLRRC69。对该基因的组织分布分析表明, CgLRRC69 mRNA在血淋巴细胞、鳃、肌肉、外套膜、心脏、消化腺和性腺中广泛表达。副溶血弧菌感染可以显著地刺激CgLRRC69在血淋巴细胞中表达, 并且在感染后6h达到峰值。同时, 酶联免疫吸附实验发现CgLRRC69可以特异性结合脂多糖(lipopolysaccharide, LPS), 表明它可能在免疫防御中有功能。吞噬实验结果显示, CgLRRC69重组蛋白可以显著地提高血淋巴细胞的吞噬能力; RNAi干扰CgLRRC69在牡蛎体内的表达, 显著降低了血淋巴细胞对细菌的清除能力。因此, 这些结果揭示了CgLRRC69作为一种新型模式识别受体, 可以特异性识别革兰氏阴性菌的主要成分LPS, 通过调理作用有效地清除细菌。     

Pathologic survey on the Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> (Adams and Reeve 1850) from Haeju off the western coastal Yellow Sea

Pathologic condition of Manila clam, Ruditapes philippinarum collected from Haeju off the west coast of North Korea in February and March 2007 was investigated in this study. The diagnostics included a protozoan parasite, Perkinsus olseni, the brown ring disease (BRD) caused by bacteria Vibrio tapetis and metazoan parasites Trematod and Cestod. P. olseni infection was examined using histology and Ray’s fluid thioglycollate medium (RFTM) assay along with Choi’s NaOH digestion technique. Trematode and cestode infection was also examined from the histology. A total of 140 clams with 35–45 mm in shell length (2–3 years old) were analyzed. Condition factor (CF), a ratio of the wet tissue weight to the shell dry weight, ranged from 0.445±0.074 (February) to 0.544±0.132 (March). Total body burden of P. olseni (i.e. infection intensity) ranged from 18,644±37,755 (February) to 26,933±80,611 cells/g wet tissue (March) with the prevalence of 76.7–43.3% (March) respectively. Prevalence of trematode and cestode infection in February and March varied 22.5–25.0 and 15.0–22.5% respectively. Infection intensity of V. tapetis ranged 1–2 and the prevalence was 1.4 (February) and 5.7% (March). Prevalence and infection intensity of P. olseni in clams from Haeju was comparatively lower than those of clams reported on the west coast of Korea. BRD and the other metazoan parasite infection were also relatively lower or similar to the condition of the clams reported previously. CF of Haeju clams was also relatively higher than the clams in Gyeonggi Bay and Taean, suggesting that Haeju clams had relatively better pathologic and health condition.     

Bioavailability of chromium from a used chrome lignosulphonate drilling mud to five species of marine invertebrates

The bioavailability of chromium from a used seawater chrome lignosulphonate drilling mud to three marine crustaceans, Portunus spinicarpus, Penaeus aztecus and Palaemonetes pugio, a polychaete worm, Neanthes virens, and a bivalve mollusc, Rangia cuneata, was evaluated. All five species showed an apparent accumulation of chromium during exposure to different types of mud-seawater mixtures. When the crustaceans were returned to mud-free seawater they rapidly released the accumulated chromium. Clams R. cuneata accumulated significant amounts of chromium when exposed to a substrate containing a layer of drilling mud. However, most of the chromium was released within 24 h when the clams were returned to a clean natural substrate, indicating that much of the chromium accumulated was in the form of unassimilated mud components in the digestive tract or on the gills. When the clams were exposed for 16 days to the mud aqueous fraction they accumulated up to 19 ppm chromium in their tissues. When returned to clean seawater, they released approximately half the chromium in 11 days. When exposed to the mud aqueous fraction, polychaetes Neanthes virerns accumulated less chromium than the other species studied, but retained nearly all the chromium during a 96 h depuration period. Bioavailability of chromium from a used chrome lignosulphonate drilling mud during short-term exposure is relatively low and is species dependent. Chromium associated with the soluble or fine particulate phase of the mud appears to be more bioavailable than that associated with the dense, rapidly sedimenting particles.     

Effects of polynuclear aromatic hydrocarbons on fishes and shellfish: An overview of research in virginia

This paper discusses the following topics related to polynuclear aromatic hydrocarbon pollution in estuaries: (1) the use of oysters (Crassostrea virginica), hard clams (Mercenaria mercenaria) and brackish water clams (Rangia cuneata) in residue monitoring; (2) the effects of elevated PAH residues on oyster condition; (3) three years of field studies relating PAH sediment contamination to abnormalities in fishes; and (4) laboratory bioassays for effects and bioconcentration modeling. Oysters, hard clams and Rangia have been shown, from 3 years of field studies, to be good monitors of pollution inputs as one proceeds along salinity gradients from 25‰ to 0·5‰. Effects of increased body burdens of PAHs are shown by a lowering of the oysters' condition index, as measured by lipid levels. Fishes inhabiting the Elizabeth River, VA, which is highly contaminated with PAHs, have abnormalities (cataracts, skin lesions, abnormal fins, etc.). These abnormalities have higher incidence in regions of the river where the sediments are more heavily contaminated. Laboratory studies utilizing contaminated sediments have reproduced some of the abnormalities observed in the field. Bioconcentration of PAHs from sediments has been studied with oysters and hard clams. Oysters generally accumulate three times the body burdens of clams exposed to the same suspensions. A faster depuration rate (k₂) for clams appears to be responsible for the higher equilibrium body burden of oysters.     

New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> challenged by the protozoan parasite <Emphasis Type="Italic">Perkinsus olseni</Emphasis>

Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.     

文蛤外套膜"肥大症"的组织病理学研究

2003年3～5月,山东莱州某养殖场部分养殖文蛤（Meretrix meretrix Linnaeus）出现外套膜异常肥大现象.组织病理学研究显示,患病贝外套膜、水管、鳃、足等器官组织内粘液细胞增多,水管两侧的外套膜结缔组织中粘液细胞增生尤为明显.同时,患病贝各器官组织还呈现出不同程度的病理学变化,主要表现为组织结构紊乱,上皮细胞肿胀、脱落,生殖细胞滞育、退化,肌纤维变形、溶解,血细胞增生并诱发炎症反应等.在患病贝体内主要观察到3种寄生性原生动物,分别为缘毛类纤毛虫、粘孢子虫及1种顶复门类“孢子虫”.纤毛虫数量较少,主要寄生于鳃表面;粘孢子虫数量较多,寄生在水管、外套膜、鳃、性腺、消化盲囊、血淋巴等器官组织中,以水管结缔组织内数量最多;而顶复门类“孢子虫”主要发现于消化道结缔组织中.组织病理学研究表明,这3种寄生虫可能是病害发生的重要原因.     

Infection by gymnophallid metacercariae enhances predation mortality of SW Atlantic stout razor clam Tagelus plebeius

Parasite life cycles are frequently completed in different hosts, thus the parasites have its life cycle overlapped to natural trophic webs. The family Gymnophallidae (Class: Trematoda; Subclass: Digenea) includes digenetic parasites whose larval stages occur on bivalves and may affect bivalve predation by the final host of these parasites. In this work we evaluated: (a) if individuals of the razor clam Tagelus plebeius with higher parasite intensity suffer higher predation by the oystercatcher Haematopus palliatus and, (b) if there is any effect of parasite intensity on burrowing and escape behaviours of these razor clams which may enhance exposure to predators. Field experiments (oystercatcher exclusion vs. open access) showed that clams with higher parasite intensity support higher predation by oystercatchers, which suggests a higher consumption of more parasitized clams and thus, a more successful reproduction of parasites linked to the intensity of infection. However, clam burrowing and escape behaviours did not show differences related to different parasite intensity, suggesting that the commonly believed mechanisms are not responsible in this case.     

温度对厚壳贻贝(Mytilus coruscus)鳃组织纤毛结构及摄食率的影响机制研究

纤毛是着生在贻贝鳃上感知外界物理和化学信号并帮助食物转运的一类重要细胞结构, 温度的变化会改变纤毛的形态和生理生化特征, 从而影响贻贝的摄食行为。为探究不同温度下厚壳贻贝的摄食率变化, 以及温度对纤毛的形态、酶活和相关基因表达的影响, 实验设置16、26和32 °C三种温度处理厚壳贻贝, 处理24 h后测定1 h内每个处理组的摄食率, 同时采用组织学方法对各处理组鳃上的纤毛形态结构进行观察,并测定鳃组织的抗氧化酶活力和纤毛相关基因tekt1、tekt2、tekt3、tekt4、caf58、caf100、dnah、rsph99和ift74的表达变化。实验结果显示, 32 °C处理组厚壳贻贝的摄食率显著低于其他两组(P<0.05), 且32 °C处理组贻贝鳃上的前纤毛、侧纤毛以及纤毛细胞受损严重并部分脱落, 血腔间隙破损暴露, 而其余两组鳃上纤毛形态较为正常; 不同温度处理下鳃组织的过氧化氢(H₂O₂)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活力呈现温度特异性; 与16 °C处理组相比, 26 °C处理组中除tekt4和ndah外其他7个纤毛相关基因表达均显著上调, 32 °C处理组除tekt1外其余8个基因表达均显著下调(P<0.05)。上述结果表明, 32 °C高温处理对贻贝鳃上纤毛造成严重损伤, 导致纤毛运动、纤毛再生和纤毛感知功能均受到一定程度的抑制,从而影响食物颗粒的运输, 造成摄食率的降低。     

三疣梭子蟹含硒谷胱甘肽过氧化物酶基因克隆及其表达分析

为探究三疣梭子蟹的免疫机制以及谷胱甘肽过氧化物酶(GPx)在甲壳动物响应寄生虫感染免疫过程中的作用,本研究采用RACE方法从三疣梭子蟹中克隆获得硒半胱氨酸-谷胱甘肽过氧化物酶基因的全长cDNA序列,其cDNA序列全长为696bp,5′-UTR长度为102bp,3′-UTR长度为87bp,开放阅读框长度为507bp,编码168个氨基酸,其中含有一个典型的由蛋白石终止密码子(220TGA222)编码的硒半胱氨酸(40U)。预测了该基因编码的氨基酸序列及其中的保守结构域,包括GPx家族签名序列(64LAFPCNQF71)、活性位点序列(152WNFEKF157)以及与酶催化活性相关的氨基酸位点包括谷氨酰胺(74Q)、精氨酸(90R和168R)和色氨酸(142W)。相似性比对和系统发育分析结果表明,三疣梭子蟹硒半胱氨酸-谷胱甘肽过氧化物酶(SeGPx)与甲壳动物中的SeGPxs相似性较高,其中与拟穴青蟹SeGPx相似性最高,并与其在系统发育树中聚为一支。经血卵涡鞭虫侵染后(0—192h),SeGPx基因在三疣梭子蟹的血细胞、肝胰腺和鳃组织中的转录水平均发生显著性升高。该结果表明,SeGPx在三疣梭子蟹应对血卵涡鞭虫的免疫反应中发挥重要作用,可通过调控甲壳宿主体内被病原扰乱的氧化还原状态进而起到宿主组织保护作用。