Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.

Regulation of the Edwardsiella tarda Hemolysin Gene and luxS by EthR

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when Fur(Et) was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Isolation, antimicrobial activity, and metabolites of fungus Cladosporium sp associated with red alga Porphyra yezoensis

Cladosporium sp. isolate N5 was isolated as a dominant fungus from the healthy conchocelis of Porphyra yezoensis. In the re-infection test, it did not cause any pathogenic symptoms in the alga. Twenty-one cultural conditions were chosen to test its antimicrobial activity in order to obtain the best condition for large-scale fermentation. Phenylacetic acid, p-hydroxyphenylethyl alcohol, and L-beta-phenyllactic acid were isolated from the crude extract as strong antimicrobial compounds and they are the first reported secondary metabolites for the genus Cladosporium. In addition, the Cladosporium sp. produced the reported Porphyra yezoensis growth regulators phenylacetic acid and p-hydroxyphenylacetic acid. No cytotoxicity was found in the brine shrimp lethality test, which indicated that the environmental-friendly Cladosporium sp. could be used as a potential biocontrol agent to protect the alga from pathogens.

Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur(vh)) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and could complement a mutant of Fur(Ec). Like Fur(Ec), Fur(Vh), possesses two cysteine residues at positions 92 and 95, yet unlike Fur(Ec), in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur(Vh) proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur(vh) are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur(Vh) are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur(Vh) is possibly different from that of Fur(Ec); and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur(Vh); and (iii) provided insights into the potential function of the local structure involving C137 and K138.

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Slat 0, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10(i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naive fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

黄海纤毛虫的丰度和生物量

海洋浮游纤毛虫是在海洋中浮游生活的一类单细胞原生动物，主要是指寡毛类纤毛虫(Oligotrich ciliates)，隶属原生动物界(Protozoa)、纤毛门(Ciliophora)、寡毛纲(Oligotrichea)，分属于Oligotrichina 和Tintinnina两个亚纲。它们个体微小，粒径在5-200 µm之间，是微型浮游动物和海洋微食物环(Marine Microbial Food Web)的重要组成部分。 2006年4月至2007年12月，在黄海(包括胶州湾)采样分析海洋浮游纤毛虫的种类组成(砂壳纤毛虫)、丰度和生物量，分析纤毛虫在这一海区的季节变化和空间变化。 纤毛虫丰度和生物量的研究方法为：Rosette采水器(胶州湾用Niskin采水器)采集水样，取1 L水样，加Lugol’s试剂固定(终浓度1%)，Utermöhl方法100倍镜检。测量虫体的体长、体宽，按最接近的几何形状(圆柱体、球体和圆锥体)计算体积。生物量由体积乘转换系数(0.19 pgC/µm3)得到，砂壳纤毛虫的肉体体积按照壳体积的1/3近似。 本文的结果表明，胶州湾各站纤毛虫平均丰度于6月达到全年最高值6065 ind./L，12月为全年丰度最低值843 ind./L。平均生物量8月达全年最高值(18.5 µg C/L)，6月为全年最低值(0.6 µg C/L)。砂壳纤毛虫种丰富度于8月达到最高值，共发现25种砂壳纤毛虫，1月种类最少(6种)。湾内站位的纤毛虫平均丰度比湾外的高(6月和8月除外)。砂壳纤毛虫在纤毛虫总丰度中的比例较小，平均为25%，范围为8-57%，分别于1月和8月达到最低和最高值。 两次冷水团大面调查结果表明，4月表层纤毛虫平均丰度(1490 ind./L)要高于10月(972 ind./L)。10月表层纤毛虫生物量0.14-5.33 µg C/L，14194站、15694站和15894站生物量较高，为4.08-5.33 µg C/L。无壳纤毛虫优势种Laboea strobila在两个航次中均呈现斑块分布，4月航次丰度0-10000 ind./L，10月航次丰度11-350 ind./L；砂壳纤毛虫优势种Ptychocylis obtusa仅在4月航次发现，最大丰度2895 ind./L，10月航次未发现。4月航次砂壳纤毛虫有百乐拟铃虫(Tintinnopsis beroidea)，丰度为0-1920 ind./L；卡拉直克拟铃虫(Tintinnopsis karajacensis)，丰度很小(10-93 ind./L)。10月航次砂壳纤毛虫优势种Tintinnidium primitivum，丰度为35-700 ind./L；也出现了尖底类瓮虫(Amphorellopsis acuta)和网纹虫(Favella spp.)，但丰度不大(0-210 ind./L)；运动类铃虫(Codonellopsis mobilis)、筒状拟铃虫(Tintinnopsis tubulosoides)和Eutintinnus sp.丰度也较低(35-105 ind./L)；Craterella torulata丰度为0-120 ind./L，主要分布于15694站。10月航次已经出现了温跃层，位于30 m左右水层，纤毛虫主要分布于温跃层之上。 六次黄海断面航次表明：温跃层在5月已经出现，到12月消失。在有温跃层的5月、6月、8月、9月，纤毛虫主要分布于温跃层(30 m左右)之上。其中8月份航次纤毛虫丰度最高，表层平均丰度3103 ind./L。12月份纤毛虫丰度最低，表层平均丰度406 ind./L。纤毛虫生物量春夏季为0.02-5.5 µg C/L，冬季为0.04-1.99 µg C/L。小型无壳纤毛虫占优势，砂壳纤毛虫东方拟铃虫(Tintinnopsis orientalis)、筒状拟铃虫、运动类铃虫、Craterella torulata和Tintinnidium primitivum几乎在各个航次均有分布。

荧光假单胞菌抗铜基因簇的表达调控及其致病性分析

CopRS/CopABCD是细菌用以维持铜内环境稳定的一个系统，虽然已在荧光假单胞菌(Pseudomonas fluorescens)中发现了CopRS/CopABCD系统的同源物，但其潜在的功能还未知。本实验在一个鱼类致病菌P. fluorescens(TSS)中鉴定到了一个基因簇，由copR、copS、copC和copD组成，但缺乏copAB。copR、copS、copC和copD基因的敲除实验发现copRSCD基因簇与TSS抗铜性相关，而且copRS操纵子和copCD操纵子在转录水平上受亚抑制水平的铜诱导。双元调控系统中的调控蛋白CopR不仅激活copCD表达，而且还激活copRS的表达。凝胶滞缓实验显示CopR能直接与copCD和copRS的启动子区域结合。干扰copR的正常表达不仅影响细菌的生长，而且还影响到细菌生物膜的形成、对鱼的侵染力和在组织中的存活力。本实验还筛选到一个CopR的突变体C104，该突变体因缺失N端的信号接受域而成为一个具有组成性活性的调控蛋白，C104在TSS中表达时导致菌株的毒力降低。本实验所发现的P. fluorescens CopR与细菌致病力之间的关系以前未见报道。

潮间带纤毛虫原生动物和小型底栖动物生态学研究

微型和小型底栖动物是底栖微/小食物网的重要构成。相对浮游生态系统, 迄今国际间对底栖食物网的认知极为欠缺。这一方面是由于微型生物形态和功能上的复杂性和多样性, 另一方面原因在于研究方法的障碍—主要是微型和小型底栖动物的定量提取和定性分析。本研究首先进行了方法学改良, 并应用新方法对底栖微食物网的重要功能类群—纤毛虫原生动物和小型底栖动物进行了不同生境的周年按月采样, 定性及定量研究的同时, 联系环境因子对微型和小型底栖生物的环境监测进行了探讨。 微型和小型底栖生物的定量研究首先涉及到目标生物在沉积物中的有效提取, 目前硅胶液提取是普遍使用的方法, 其中Ludox液主要应用于小型生物, 它不但价格便宜而且比重合适, 因此在常规生态研究中被广为接受。不过, Ludox易与于海水中的阳离子产生凝结而无法直接用于微型生物; 目前唯一直接应用于微型生物提取的是Percoll硅胶液, 但其昂贵的价格使其在常规生态研究中受到极大限制。本研究以价格低廉的Ludox 硅胶液结合定量蛋白银染色 (QPS) 技术开发了一种新的方法, 即Ludox-QPS法。主要流程为: 样品采集与固定、淘洗/稀释降盐、Ludox密度梯度离心、过滤浓缩和琼脂包埋, QPS染色、永久封片及鉴定计数。添加已知数量的纤毛虫至无生物底泥的重获实验表明, 该密度梯度离心的提取率大于94%; 该方法对自然沉积物中纤毛虫的提取率达97.6%, 对沙质、泥沙质和泥质中海洋线虫的提取率分别达97%、96.9% 和97.8%。对比实验表明, 经QPS制片获得的小型动物的丰度和类群数量与传统方法相当或更高, 尤其当小个体虫体占优势时, 该法显示出较传统方法 (导致数量低估) 更为明显的定量优越性。该方法除用于纤毛虫和小型动物的定量分析外, 还具有较高的分类分辨率, 染色后的纤毛虫原生动物大多类群可鉴定到属, 部分可鉴定到种, 以此可在群落水平上研究其生态作用。 根据新开发的Ludox-QPS技术, 在大沽河潮间带依据盐度梯度选定2个站位 (IIQ和营海) 进行了周年按月采样, 对底栖纤毛虫和小型底栖动物进行了定量研究。纤毛虫原生动物在IIQ和营海的年平均丰度分别为2236 inds./10 cm2 和935 inds./10 cm2 (28 inds./ml 和12 inds./ml), 平均生物量分别为119.1 gC/10 cm2和54.2 gC/10 cm2 (1.5 gC /ml 0.7 gC/ml)。丰度的季节变化趋势为: 春天 > 秋天 > 夏天 > 冬天。垂直分布上, 在营海分布于表层0-0.5 cm 的比例为57.1%, 分布于0.5-2 cm、2-4 cm和4-8 cm比例分别为23.1%、11.4% 和8.5%; 13个月中除12月份外, 4-8 cm均有一定数量的纤毛虫分布; 而在IIQ, 97% 的纤毛虫分布在0-0.5 cm, 分布在0.5-2 cm、2-4 cm和4-8 cm比例分别为2.4%、0.4%和0.2%, 4-8 cm的分布只发生在春季和秋季。纤毛虫的多样性季节变化明显, 春秋季物种丰富, 两个站点每毫升沉积物的平均物种数分别为18和6。Two-Way Crossed ANOSIM 分析表明纤毛虫群落在月份间和站点间的差异极其显著。Pseudochilodonopsis sp., Chilodontopsis sp., Euplotes sp.及Prorodon sp.是表征两个生镜中纤毛虫群落的主要类群。 同时, 发现了14个小型生物类群, 其中线虫在IIQ和营海的丰度优势度分别为97.4% 和78.6%。小型动物在IIQ和营海的年平均丰度分别为4793 inds./10 cm2和8915 inds./10 cm2 (60 inds./ml和111 inds./ml), 其生物量分别为1068.8 gC /10 cm2和1790 gC /10 cm2 (13.4 gC/ml和22.4 gC/ml)。小型底栖动物的丰度在IIQ的季节变化为: 夏季 (7888 inds./10cm2) > 秋季 (5447 inds./10cm2) > 春季 (3731 inds./10cm2) > 冬季 (2780 inds./10cm2); 在营海则完全相反: 冬季 (15579 inds./10cm2) > 春季 (10691 inds./10cm2) > 秋季 (6611 inds./10cm2) > 夏季 (4667 inds./10cm2)。小型底栖动物和纤毛虫的相对重要性存在明显的区域和季节差异。 纤毛虫原生动物、小型动物及环境因子的相关分析表明, 纤毛虫的丰度和多样性与温度和盐度及有机质含量显著相关, 与小型动物没有显著相关性; 群落结构分析表明, 温度、有机质和小型动物的丰度的组合与纤毛虫群落丰度的相关系数为0.345; 盐度、脱镁叶绿素、有机质和小型动物生物量的组合与纤毛虫群落多样性的相关系数为0.403。依据海洋线虫和桡足类的比值 (N/C) 推测, IIQ 可能存在严重的有机污染, 营海则存在明显的季节波动, 8月和9月及2月可能是污染最严重的季节, 这种状况在纤毛虫群落结构的CLUSTER聚类中得到验证。虽然目前尚没有形成有关微型底栖生物-纤毛虫原生动物的污染检测的直接依据, 但本研究说明纤毛虫群落的确对环境污染具有一定的感应度, 而且这种感应和利用小型生物的主要类群估算的污染检测 (N/C) 存在一定程度的关联。 90年代早期有关青岛湾有机污染带的研究表明, 经彻底截污后, 其环境状况向良性发展。进一步了解该湾的健康状况, 2006.5-2007.5月对该湾沙质和泥沙质的小型动物进行周年按月采样。小型动物在泥沙质和砂质沉积物中的年平均丰度分别为4853 ± 1292 inds./10 cm2和1528 ± 569 inds./10 cm2; 年平均总生物量分别为1434.1 ± 897.0 gC /10cm2和720.7 ± 353.8 gC/10cm2。沙质底小型生物的丰度季节波动明显, 6月份和12月份最高, 3月份和9月份最低; 泥沙质季节波动不明显, 6月份最高。两个站点均有48%的小型动物分布在0-0.5 cm 表层, 海洋线虫在表层的分布比例分别为48% (泥沙质) 和34% (砂质)。共检获14个小型动物类群, 其中线虫在泥沙质和砂质沉积物中的年平均丰度分别4619 ± 1255 inds./10cm2和1014 ± 376 inds./10cm2, 其丰度优势度分别为95.2%和66.4%。其它在丰度上占优势的类群, 泥沙质依次为多毛类 (1.5%)、甲壳幼体 (1.5%) 和桡足类 (0.7%); 沙质依次为: 甲壳类幼体 (12.6%)、腹毛类 (8.3%) 和 桡足类 (6.2%)。CLUSTER聚类分析表明, 泥沙质和和砂质中小型生物的丰度组成具有64%的相似性。BIOENV分析表明, 温度、盐度、中值粒径和粘土粉砂含量的组合最能解释不同月份之间和不同站位间的差异, 其相关系数为0.614。依据小型生物的丰度和类群组成, 表明泥沙质底尚存一定的有机污染。

A bioaugmentation failure caused by phage infection and weak biofilm formation ability

Two biological aerated filters (BAF) were setup for ammonia removal treatment of the circulation water in a marine aquaculture. One of the BAFs was bioaugmented with a heterotrophic nitrifying bacterium, Lutimonas sp. H10, where the ammonia removal was not improved and the massive inoculation was even followed by a nitrification breakdown from day 9 to 18. The nitrification was remained stable in control BAF operated under the same conditions. Fluorescent in situ hybridization (FISH) with rRNA-targeted probes and cultivable method revealed that Lutimonas sp. H10 almost disappeared from the bioaugomented BAF within 3 d, and this was mainly due to the infection of a specific phage as revealed by flask experiment, plaque assay and transmission electron observation. Analyses of 16S rRNA gene libraries showed that bacterial groups from two reactors evolved differently and an overgrowth of protozoa was observed in the bioaugmented BAR Therefore, phage infection and poor biofilm forming ability of the inoculated strain are the main reasons for bioaugmentation failure. In addition, gazing by protozoa of the bacteria might be the reason for the nitrification breakdown in bioaugmented BAF during day 9-18.

Summertime ciliate and copepod nauplii distributions and micro-zooplankton herbivorous activity in the Laizhou Bay, Bohai Sea, China

The abundance and biomass of ciliated protozoa and copepod nauplii were investigated at 21 grid stations and two anchored stations in the Laizhou Bay, Bohai Sea, China in June 1998. Dilution incubations were carried out to investigate micro-zooplankton grazing pressure at the anchored stations during spring tide and neap tide. The dominant species were Tintinnopsis amoyensis, T. chinglanensis, T. pallida and aloricate ciliates. A total of 13 species of tintinnids were found. The total abundance of ciliates and nauplii ranged from 30 to 2390 ind l(-1) at grid stations. Tintinnopsis amoyensis was the only ciliate found at the anchored stations and in concentrations which varied from 0 to 6700 ind l(-1). The spatial distribution of ciliates was patchy. Tintinnopsis amoyensis and T. pallida were distributed in the Weihe River mouth and Xiaoqinghe River mouth respectively. The aloricate ciliates, T. chinglanensis and Codonellopsis ostenfeldi dominated offshore in sequence. The water mixing process may affect the spatial pattern of the dominant ciliate species. The abundance and biomass of copepod nauplii were in the range of 0-140 ind l(-1) and 0-7 mu g C l(-1) respectively, with the peak appearing at grid station 15. The total biomass of ciliates and copepod nauplii was in the range of 1(.)5-25 mu g C l(-1). Water column biomass of ciliates and nauplii varied from 2(.)37 to 52(.)3 mg C m(-2). At the anchored stations, the phytoplankton growth rates ranged from undetectable to 0 21 d(-1) and micro-zooplankton grazing rates from 0 13 to 0(.)57 d(-1). The grazing pressure of micro-zooplankton were 12 to 43% of the chlorophyll standing stock and 84 to 267% of the chlorophyll (C) 2000 Academic Press.