胶州湾浮游病毒的分布研究

运用荧光显微镜技术,对2007年6～8月胶州湾14个站点的浮游病毒丰度进行了检测,分析了病毒在不同月份的水平与垂直分布变化,发现胶州湾浮游病毒的丰度在0.48×107～22.78×107个/mL之间,平均值为(5.72±4.72)×107个/mL,7月份病毒丰度明显高于其他两个月(P0.01)。病毒呈现从湾内至湾口至湾外递减的趋势,病毒垂直分布变化不明显。病毒-细菌比率(VBR)范围为3.90～150.72,平均值42.05±28.55,处于较高水平。利用多元相关性分析发现,病毒丰度与异养细菌丰度、聚球藻蓝细菌丰度和叶绿素a含量相关,其相关系数r分别为0.605(P0.01),0.265(P0.01)和0.604(P0.05),确定系数R2分析表明,异养细菌和叶绿素a对浮游病毒丰度的影响基本相当。病毒丰度与温度、盐度无明显相关性。对VBR的分析表明,调查区域藻类病毒占总浮游病毒的比例较高;通过VBR与异养细菌丰度的负相关性分析,认为胶州湾噬菌体的宿主菌种群较单一。     

冬季和春季长江口及其近海水域浮游病毒丰度的分析

采用荧光显微技术,对2006年长江口及近海水域20个站点的表层及10m层或潜水体冬、春两季的浮游病毒丰度进行了检测,对浮游病毒丰度在季节（冬、春两季）、水平分布和垂直分布上的变化进行了探讨.调查区浮游病毒丰度在冬、春季节上并无明显差异,但在水平分布上存在很大差异,河口区浮游病毒直接检测量（Virus Direct Count, VDC）达到10^7个/ml,近海水域VDC为10^6个/ml,河口区的浮游病毒丰度都明显高于近海水域病毒丰度 （P＜0.01）.在垂直分布上,冬、春两季长江口水域水深小于10m的站位,表层浮游病毒丰度与底层病毒丰度无明显差别,水深大于10m的站位,表层水样的浮游病毒丰度都高于10m水层病毒丰度,说明长江口浮游病毒的垂直分布与站位总水深有关.还通过比较各站点VDC与叶绿素a含量的数据,分析了二者之间的相关性：冬季浮游病毒丰度与叶绿素a含量成正相关性;春季浮游病毒丰度与叶绿素a含量成负相关性,但病毒丰度受叶绿素a含量的影响仅为10%-11%.     

International accreditation of sandwich hybridisation assay format DNA probes for micro‐algae

The sandwich hybridisation assay (SHA) is a DNA probe‐based method for rapid identification and enumeration of toxic micro‐algae which uses species specific oligonucleotide probes targeted at ribosomal RNA. It is suited to fragile micro‐algal cells which commonly collapse during the fixation stage of sample collection, compromising identification by traditional microscopy. The assay has been available for research for several years, but was validated and accepted for international accreditation for commercial laboratory use in New Zealand in May 2004 (International Accreditation New Zealand: ISO 17025). During the validation of the raphidophyte assay, some discrepancies were noted between SHA cell concentration estimates and traditional light microscope cell counts. Higher SHA estimates were recorded when blooms had collapsed but rRNA was still present in sea water. Conversely, higher traditional cell counts occurred when sample delivery was delayed more than 48 h, presumably owing to degradation of rRNA in the live cultures used for the SHA. SHA cell concentration estimates of the toxic diatom bloom‐former Pseudo‐nitzschia australis were also compared with whole cell format DNA probe counts and traditional microscope counts; SHA counts were comparable for the three methods tested.     

Effects of fixation and storage on flow cytometric analysis of marine bacteria

Flow cytometry (FCM) is now becoming a routine tool for the enumeration and optical characterization of bacteria in marine environments. We investigated the effects of sample fixation and storage upon flow cytometric determination of marine bacteria. Fixed and unfixed seawater samples were analyzed by FCM immediately aboard ship and/or later in the laboratory, and the appearances of the fluorescence signals and bacterial counts of these samples were compared. Fixation and storage led to the formation of multiple peaks in fluorescence histograms; this was also seen in 22 out of 36 samples frozen in liquid nitrogen. Fixation did not, but storage did induce a decrease of bacterial counts: a rapid decrease during the first 3 days followed by a slower decline. The decline of cell numbers in stored samples was expressed by a regression model. Our studies indicate that precaution is necessary when interpreting the data from fixed and/or stored marine bacterial samples analyzed by FCM. The possibility that the procedure of fixation and storage leads to the appearance of high DNA and low DNA bacterial groups should be considered.     

Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picophytoplankton: Fixation and Storage Effects

The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics.     

海洋浮游植物病毒的研究进展

海洋浮游植物病毒在海洋生态系统中,特别是在赤潮的消亡过程中起了很重要的作用。介绍了海洋浮游植物病毒的种类、多样性和丰度分布及其在海洋生态系统中的主要作用和研究现状。重点介绍和比较了海洋浮游植物病毒多样性、丰度的DNA聚合酶链扩增法(PCR)、透射电镜法、荧光显微镜法和流式细胞仪法等研究方法及其发展状况,以期为今后的研究奠定基础。     

Responses of phytoplankton and heterotrophic bacteria in the northwest subarctic Pacific to in situ iron fertilization as estimated by HPLC pigment analysis and flow cytometry

To verify the hypothesis that the growth of phytoplankton in the Western Subarctic Gyre (WSG), which is located in the northwest subarctic Pacific, is suppressed by low iron (Fe) availability, an in situ Fe fertilization experiment was carried out in the summer of 2001. Changes over time in the abundance and community structure of phytoplankton were examined inside and outside an Fe patch using phytoplankton pigment markers analyzed by high-performance liquid chromatography (HPLC) and flow cytometry (FCM). In addition, the abundance of heterotrophic bacteria was also investigated by FCM. The chlorophyll a concentration was initially ca. 0.9 μg l⁻¹ in the surface mixed layer where diatoms and chlorophyll b-containing green algae (prasinophytes and chlorophytes) were predominant in the chlorophyll biomass. After the iron enrichment, the chlorophyll a concentration increased up to 9.1 μg l⁻¹ in the upper 10 m inside the Fe patch on Day 13. At the same time, the concentration of fucoxanthin (a diatom marker) increased 45-fold in the Fe patch, and diatoms accounted for a maximum 69% of the chlorophyll biomass. This result was consistent with a microscopic observation showing that the diatom Chaetoceros debilis had bloomed inside the Fe patch. However, chlorophyllide a concentrations also increased in the Fe patch with time, and reached a maximum of 2.2 μg l⁻¹ at 5 m depth on Day 13, suggesting that a marked abundance of senescent algal cells existed at the end of the experiment. The concentration of peridinin (a dinoflagellate marker) also reached a maximum 24-fold, and dinoflagellates had contributed significantly (>15%) to the chlorophyll biomass inside the Fe patch by the end of the experiment. Concentrations of 19′-hexanoyloxyfucoxanthin (a prymnesiophyte marker), 19′-butanoyloxyfucoxanthin (a pelagophyte marker), and alloxanthin (a cryptophyte marker) were only incremented a few-fold increment inside the Fe patch. On the contrary, chlorophyll b concentration reduced to almost half of the initial level in the upper 10 m water column inside the Fe patch at the end of the experiment. A decrease with time in the abundance of eukaryotic ultraphytoplankton (<ca. 5 μm in size), in which chlorophyll b-containing green algae were possibly included was also observed by FCM. Overall, our results indicate that Fe supply can dramatically alter the abundance and community structure of phytoplankton in the WSG. On the other hand, cell density of heterotrophic bacteria inside the Fe patch was maximum at only ca. 1.5-fold higher than that outside the Fe patch. This indicates that heterotrophic bacteria abundance was little respondent to the Fe enrichment.     

Effects of virus infection on expression of cell cycle regulatory proteins in the unicellular marine algae Emiliania huxleyi

The authors have investigated the biochemical events by which marine algal virus infection induces cell cycle arrest. The key G₂/M-phase regulatory proteins are analyzed by immunobloting in unicellular Emiliania huxleyi, suggesting that virus induced cell cycle arrest is related with virus''s effect on cyclins and cyclin dependent kinases. E. huxleyi virus (EhV) represses Cdc2/cyclinB complex activity by inhibiting the activity of Cdc2 kinase in a phosphorylation-related manner, blocking host cells G₂/M checkpoint. Dephosphorylated/inactive Cdc25C combined with up-regulation of Wee1 expression at early infect period appears to be important mechanisms by which EhV represses Cdc2/cyclinB complex activity that is required for entry into M phase. This study has allowed us to confirm that algal virus infection leads to selective activation or inhibition of certain cell-cycle factors, which may play a significant role in establishing a more efficient environment for viral gene expression and DNA replication.     

应用DGGE技术分析北黄海和青岛近海藻类DNA病毒遗传多样性

采用DGGE技术对北黄海和青岛近海藻类DNA病毒群落多样性进行调查。结果表明,在北黄海A604站、B107站和C803站分别得到10个、8个、8个OTUs,在青岛近海ZQ站得到6个OTUs,说明不同海域藻类DNA病毒多样性不同,在北黄海较丰富,青岛近海较低。北黄海3个站间相比,OTUs数量和位置均存在差异,表明同一海域不同站位藻类DNA病毒多样性也存在差异。在所得OTUs中,有2个OTUs在4个站中均存在,说明在北黄海和青岛近海存在着一些共同的藻类DNA病毒。4个站点藻类DNA病毒优势种均不相同,在A604站和ZQ仅有一个优势种,而在B107和C803各有两个优势种。     

Modification of the polyethylene glycol 6000 precipitation method for recovering human and indicator viruses from oysters and mussels

Human viruses are a common contaminant of shellfish affected by human sewage wastes. They are difficult to detect as they are not easily separated from shellfish tissue. This paper describes a modification of the polyethylene glycol (PEG) precipitation technique for recovery of enteroviruses and F‐specific bacteriophages from the Pacific oyster (Crassostrea gigas) and the green‐lipped mussel (Perna canaliculus). Modifications adopted were the use of only the digestive gland tissue for virus extraction, resuspension of the primary PEG pellet in 4 volumes of eluent, and the introduction of a secondary PEG precipitation to reconcentrate the virus containing extract. The recovery rate of the virus extraction process was not affected by introduction of the secondary concentration step (overall recovery remained at 60–70% of the virus input). The advantages of reduction of tissue residue in the extract, smaller final volume, and the ability to process 2–3 times the number of individual shellfish for the same effort, improve the practicality of the method.     

Effects of sea urchin (Evechinus chloroticus) grazing in Dusky Sound,Fiordland, New Zealand

Evechinus chloroticus (Val.) is known to be an important grazer of subtidal barren habitats in northern New Zealand but its role in structuring rocky reef communities at colder southern localities is poorly understood. The present study was prompted by a proposal for the establishment of an E. chloroticus fishery in Dusky Sound, Fiordland, New Zealand. To determine the ecological importance of E. chloroticus in Dusky Sound, sea urchins were experimentally removed from shallow fucoid fringe (upper zone), mid‐depth barrens (middle zone), and deeper algal meadow (lower zone), at eight sites and compared with a paired set of eight control sites. Two years of continuous removal of E. chloroticus resulted in conspicuous changes to the algal community at all depth zones examined. The density of fucoid algae (Carpophyllum spp., Sargassum spp., Cystophora spp., and Landsburgia quercifolia) increased mostly in the upper and middle zones, although smaller increases were also observed in the lower zone. Changes in cover of herbaceous, turfing and crustose algae observed in the middle and lower zones were larger than in the upper zone. Density increases of the kelp Ecklonia radiata were observed mainly in the lower zone, although still important in the middle zone. These results show that E. chloroticus can have a strong influence on the structure of algal assemblages in Dusky Sound hence the development of an E. chloroticus fishery in this area should be treated with caution.     

Genetic and meristic variation in black and smooth oreos in the New Zealand exclusive economic zone

Meristic and genetic methods were used to determine the stock relationships of black Allocytus niger (James, Inada & Nakamura, 1988) and smooth oreo Pseudocyttus maculatus (Gilchrist, 1906) in the New Zealand Exclusive Economic Zone (EEZ). Samples were collected from four management areas—OEO 1 (south‐west), OEO 3A (Chatham Rise west), OEO 4 (Chatham Rise east), and OEO 6 (subantarctic) during the 1998 October‐December spawning period. Lateral line scale counts and pyloric caeca counts revealed differences between black oreo samples from OEO 6 and the other three OEO management areas. Lateral line scale counts in smooth oreo showed no significant differences between areas. Genetic analyses of four non‐coding regions of nuclear DNA and mitochondrial (mt) DNA haplotypes in black oreo showed no significant differentiation among the four management areas. Likewise for smooth oreo, genetic analyses of five non‐coding regions of nuclear DNA and mtDNA haplotypes showed no overall regional differentiation, although there was weak evidence at one locus for a difference between OEO 3A and OEO 6. The data do not reject the null hypothesis of a single genetic stock in New Zealand waters, and are typical of marine species with long pelagic juvenile stages.     

The response of the virus community to the SEEDS II mesoscale iron fertilization

Although the important role of viruses in marine biogeochemical cycles has been established in recent years, virus activity (including changes in this activity) has been largely ignored during mesoscale iron (Fe)-fertilization experiments relative to other processes. This is of particular interest as viruses have been shown to be critical to the transformation of Fe from the particulate (i.e., biological) to the dissolved pools. The goal of the present study was to evaluate changes in the virus-mediated lysis of heterotrophic bacterial cells following a shift in ecosystem trophic status brought about by a mesoscale Fe addition in the subarctic Pacific Ocean. Virus production rates, estimated by a reduction and reoccurrence assay, were coupled with transmission electron microscopy estimates of burst size and direct counts of virus and bacterial abundance. Fe fertilization of the upper mixed layer resulted in significant yet weak increases in virus production rates during the 12 days of observation immediately after fertilization, although the burst size (viruses produced per lytic event) and the percentage of visibly infected cells remained constant. The results imply that increases in virus production rates were most likely tied to a decreased lytic cycle length or the stimulation of lysogenized cells following the stimulation of primary and secondary productivity by the addition of Fe. The results also indicate that virus-induced cell-lysis regenerated an estimated nearly 200 pmol L⁻¹ Fe daily, providing a significant return of Fe back to the water column, which may be critical in the maintenance of this added Fe as resident.     

Loss Processes of Sinking Fecal Pellets of Zooplankton in the Mesopelagic Layers of the Antarctic Marginal Ice Zone

A time-series sediment trap deployment was carried out in the marginal ice zone (MIZ) of the Antarctic Ocean (64°42′ S, 139°58′E; sea depth of 2930 m), during the austral summer. Cylindrical fecal pellets were the predominant sinking particles at 537 m in the middle of January and most of them disappeared below that depth, the loss of which were 25.3 mg C m⁻² day⁻¹ in the depth range of 537–796 m. Small-sized sinking particles other than fecal pellets increased in that depth range. Analyses of fecal pellets for remnant DNA corresponding to 16S mitochondrial RNA and 28S ribosomal RNA suggested that the large cylindrical fecal pellets at 537 m were produced by Antarctic krill (Euphausia superba) and copepods. According to the presence of the DNA associated with sinking particles, E. superba fecal pellets rapidly disappeared below 537 m, while copepod fecal pellets still remained in the mesopelagic and bathypelagic layers. Small-sized amorphous sinking particles at 537 m also contained E. superba- and copepod-derived DNA. The abundance of trap-collected copepods (Oithona spp. and Oncaea spp.) which are known to be coprophagous increased at 796 m where many fecal pellets disappeared. We suggest that those rapidly sinking pellets were fragmented by copepods with intensified coprorhexy activity (fragmentation of fecal pellets) in the mesopelagic layers, reducing their sinking rates. These smaller and slower sinking particles can be important food sources for detritivorus or coprophagous animals in mesopelagic and bathypelagic layers in the MIZ. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of the performance of HPLC–CHEMTAX analysis for determining phytoplankton biomass and composition in a turbid estuary (Schelde, Belgium)

In the upper Schelde estuary in 2002, phytoplankton biomass and community composition were studied using microscopic and pigment analyses. Chlorophyll a concentration was a good predictor of phytoplankton biomass estimated from cell counts and biovolume measurements. The phytoplankton carbon to chlorophyll a ratio, however, was often unrealistically low (<10). CHEMTAX was used to estimate the contribution of the major algal groups to total chlorophyll a. The dominant algal groups were diatoms and chlorophytes. While diatom equivalents in chlorophyll a predicted diatom biomass relatively well, chlorophyte equivalents in chlorophyll a were only weakly related to chlorophyte biomass. The pigment-based approach to study phytoplankton overestimated phytoplankton biomass in general and chlorophyte biomass in particular in late autumn and winter, when phytoplankton biomass was low. A possible explanation for this overestimation may be the presence of large amounts of vascular plant detritus in the upper Schelde estuary. Residual chlorophyll a, chlorophyll b and lutein in this detritus may result in an overestimation of total phytoplankton and chlorophyte biomass when the contribution of phytoplankton to total particulate organic matter is low.     

藻体部位、藻质量、碳源、水流对铁钉菜和羊栖菜营养盐吸收的影响

采用实验室生态学研究方法,进行了藻体部位、藻质量、碳源质量浓度及水流流速对铁钉菜(Ishige foliaceaokamurai)和羊栖菜(Sargassum fusiforme)氮磷营养盐吸收影响的研究。结果表明:2种大型海藻不同部位对营养盐的吸收速率不同,铁钉菜下部的吸收速率最大,其次是固着器;羊栖菜顶部的吸收速率最大,其次是中部。铁钉菜藻质量1.0g的吸收速率最大,其次是1.5g;羊栖菜藻质量0.5 g的吸收速率最大,其次是1.0g;2种藻类2.0g和2.5g的吸收速率相差不多。碳源质量浓度为1200mg/L时,铁钉菜的吸收速率最大,其次是碳源800mg/L;碳源质量浓度为800mg/L时,羊栖菜的吸收速率最大,其次是碳源400mg/L;2种海藻对照组的吸收速率均相对较低。相同流速下,2种海藻均以PO4-P对照组的吸收速率最大;NO3-N、NH4-N吸收速率在流速为50cm/s时最大。     

Discrepancy between otolith and tag-recovery estimates of growth for two South African surf-zone teleost species

Growth rates determined from recovered tagged galjoen Dichistius capensis and white steenbras Lithognathus lithognathus were compared to predictions from growth models based on otolith ring counts. Galjoen could not be sexed externally, but it was assumed that all fish >450 mm total length were females, which grow faster than males. Those smaller than this were assumed to include mostly males and were treated as a "male" sample. Male and female white steenbras grow equivalently. Tagged "male" (n = 322), and female galjoen (n = 34) and white steenbras (n = 14) grew more slowly than the model predictions. The discrepancy is likely attributable to the physiological effect of external tags on growth.     

用基因枪将GUS基因导入褐藻细胞中表达

于１９９３年１１月－１９９４年２月，用高压氦气式基因枪，将ＰＨ１２２１质粒装有ＧａＭＶ ３５Ｓ启动子，ＧＵＳ基因以及ｎｏｓ的３＇调控区导入海带和裙带菜组织切块中。４８ｈ后，在海带假根细胞和裙带菜中肋部叶片细胞中检测到ＧＵＳ基因的表达。实验表明，微粒子轰击法是外源基因导入大型褐藻的一个有效途径。ＧａＭＶ ３５Ｓ启动子能够驱动外源基因在海洋藻类中的表达，可作为藻类基因工程的启动元件。     

Effects of artificial shading on periphyton and invertebrates in a New Zealand stream

The association between abundance of invertebrates and presence of extensive periphyton cover in the Waitakere River (36° 28′S, 174° 31′E), northern New Zealand, was studied from October 1974 to June 1975. A black polythene canopy (44 m²) was placed across the stream, and the quantity of algal material and numbers of invertebrates in shaded and unshaded areas was measured. The presence of the canopy and the associated absence of periphyton influenced the distribution of several invertebrate species; those more abundant beneath the canopy were Slavina appendiculata (Oligochaeta: Naididae), Austrosimulium australense (Diptera: Simuliidae), and Aoteapsyche colonica (Trichoptera: Hydropsychidae), whereas the chironomids Maoridiamesa harrisi (Diamesinae), Austrocladius sp. (Orthocladiinae), and Paratanytarsus agameta (Chironominae) and the trichopterans Hydrobiosis parumbripennis (Rhyacophilidae), Oxyethira albiceps (Hydroptilidae), and Pycnocentrodes spp. (Conoesucidae) were more abundant outside. Potamopyrgus antipodarum (Mollusca: Hydrobiidae) and Hydora nitida (Coleoptera: Elmidae), commonly recorded from algal mats in New Zealand, were equally abundant in shaded and unshaded areas. Differences in distribution between experimental and control sites may have been due to interference by periphyton with attachment sites (simuliids and hydropsychids) and feeding habits of the invertebrates (remaining species).     

改性粘土絮凝后残留的太平洋亚历山大藻(Alexandrium pacificum)毒素变化研究

改性粘土法是一种高效、环保的有害藻华应急处置技术,可通过絮凝作用有效去除水体中藻华生物。但利用改性粘土絮凝产毒藻后,水体中胞内外藻毒素的变化情况目前尚不清楚。文章考察了I型改性粘土(MCI)絮凝典型产毒甲藻——太平洋亚历山大藻(Alexandriumpacificum)后,水体中残留藻细胞内和胞外麻痹性贝类毒素(paralytic shellfish toxins, PSTs)含量、组分的变化情况。实验结果表明, MC I对密度为6.11×10~3 cells/mL的A. pacificum 3小时去除率达62%,水体中残留藻细胞单细胞毒素含量和PSTs组分与对照组无显著差异,但水体中总PSTs含量大大降低,其中由絮凝沉降导致的胞内PSTs被去除量占水体中PSTs总减少量的90%以上。另外,针对MC I对胞外PSTs吸附效果的研究发现,低于0.5 g/L的MC I对胞外PSTs无明显吸附效果,而在利用0.2 g/L MC I絮凝去除大部分亚历山大藻后,水体中胞外PSTs含量无明显变化。由此可以推测该用量下的MC I未造成大量藻细胞破裂向水体中释放毒素。该研究结果将为改性粘土治理有毒甲藻藻华的现场应用提供科学依据。