Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: stress resistance of artificially raised young seedlings revealed by chlorophyll fluorescence measurement

Commercial farming of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura in China and South Korea in the sea depends on three sources of seedlings: holdfast-derived regenerated seedlings, young plants from wild population and zygote-derived seedlings. Like many successfully farmed seaweed species, the sustainable development of Hizikia farming will rely on a stable supply of artificial seedlings via sexual reproduction under controlled conditions. However, the high rate of detachment of seedlings after transfer to open sea is one of the main obstacles, and has limited large-scale application of zygote-derived seedlings. To seek the optimal condition for growing seedlings on substratum in land-based tanks for avoidance of detachment in this investigation, young seedlings were grown in both outdoor tanks exposed directly to sunlight and in indoor raceway tanks in reduced, filtered sunlight. Results showed that young seedlings, immediately after fertilization, could withstand a daily fluctuation of direct solar irradiance up to a level of 1800 mu mol photons m(-1)s(-1), and maintained a faster growth rate than seedlings grown in indoor tanks. Detailed experiments by use of chlorophyll fluorescence measurements further demonstrated that the overnight (12 h) recovery of optimal fluorescence quantum yield (F-v/F-m) of seedlings after 1 h treatment at 40 degrees C was 98%, and the 48 h recovery of F-v/F-m of seedlings after 1 h exposure to 1800 mu mol m(-2)s(-1) was 92%. Forty-one-day-old seedlings showed no significant decrease of optimal fluorescence quantum yield at salinity ranging from 30 to 5 ppt for a treatment up to 17 h. Six-hour desiccation treatment did not have any influence on the optimal fluorescence quantum yield. Exposure to 18 mmol L-1 sodium hypochlorite for 10 min did not damage the PSII efficiency, and thus could be used to remove epiphytic algae. The strong tolerance of young seedlings to high temperature, high irradiance, low salinity and desiccation found in this investigation supports the view that mass production of Hizikia seedlings should be performed in ambient light and temperature instead of in shaded greenhouse tanks.

Zoospores of Undaria pinnatifida: their efficiency to attach under different water velocities and conjugation behavior during attachment

In the invading course of Undaria pinnatifida, zoospore attachment in a dynamically changed subtidal water environment is crucial for the establishment of a potential population in alien waters. Among many abiotic factors that may interfere with the attachment process, water velocity is the most important one. In this investigation, the effect of water velocity on zoospore attachment of U. pinnatifida was investigated in an artificially designed system. It was found that freshly released zoospores that were transported by water flowing at 0 similar to 16 cm/s showed no difficulty in attaching the smooth surface. Zoospore attachment decreased at elevated water flowing rates. At 70 cm/s no spore attachment occurred. Spores that have settled on glass slide for up to I h could not be stripped away by flowing water at a rate of 129 cm/s, the same was true of the 20 d old filamentous gametophytes. It was found that more than 70% of free-swimming zoospores tended to settle down adjacent to the settled spores and formed conjugated clusters from two up to a few hundred cells in still culture.

Circadian rhythms in the growth and reproduction of the brown alga Undaria pinnatifida and gametogenesis under different photoperiods

Circadian growth rhythm of the juvenile sporophyte of the brown alga Undaria pirznatifida was measured with the computer-aided image analysis system in constant florescent white light under constant temperature (10 degrees C). The growth rhythm persisted for 4 d in constant light with a free-running period of 25. 6 h. Egg release from filamentous gametophytes pre-cultured in the light - dark regime was evaluated for six consecutive days at fixed time intervals in constant white light and 12 h light per day. Egg release rhythm persisted for 3 d in both regimes, indicating the endogenous nature. Temporal scale of egg release and gametogenesis in 18, 16, 12 and 8 h light per day were evaluated respectively using vegetatively propagated filamentous gametophytes. Egg release occurred 2 h after the onset of dark phase and peaked at midnight. Evaluation of the rates of oogonium formation, egg release or fertilization revealed no significant differences in four light-dark regimes, indicating; the great plasticity of sexual reproduction. No photoperiodic effect in gametogenesis in terms of oogonium formation and egg release was found, but fertilization in short days was significantly higher than in long days. Results of this investigation further confirmed the general occurrence of circadian rhythms in inter-tidal seaweed species.

Antioxidant properties of recombinant allophycocyanin expressed in Escherichia coli

Allophycocyanin (A-PC) is the main core component of phycobilisome found in blue-green algae. The apo-allophycocyanin and its subunits were expressed in Escherichia coli and their antioxidant properties were evaluated using deoxyribose assay. The result showed that both recombinant allophycocyanin fused with maltose binding protein (MBP) tag and 6 x His-tag and their alpha or beta subunits can scavenge hydroxyl radicals successfully, and the separated g or beta subunits had a higher inhibition effect on hydroxyl radicals than that when they combined together. The scavenging effects increased with the increasing concentration. These results clearly suggested that apo-allophycocyanin is involved in the antioxidant and radical scavenging activity of phycocyanin, and the antioxidant activity may be partially responsible to the anti-tumor effect of the recombinant allophycocyanin. (c) 2006 Elsevier B.V. All rights reserved.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

Dynamic changes of total bacteria and Vibrio in an integrated seaweed-abalone culture system

Polyculture of seaweeds alongside fed animal aquaculture is an environmentally friendly means of avoiding eutrophication problem both in land-based and sea-based monoculture systems. Many aspects of such polyculture systems have been described, but little attention has been given to the impact of live seaweeds on the microbiological properties of the water that connects the algae and animals. In this investigation, the Pacific red alga Gracilaria textorii was cultured in a recirculated dual tank system (150 L) with the juvenile abalone Haliotis discus hannai. Dynamic changes of total bacteria (TB) and total Vibrio (TV) in the water of polyculture and monoculture systems were evaluated. Results revealed that (1) level of TB in the polyculture was constantly higher than in the monoculture over a 6.5-day period. While levels of TV in the polyculture was detected to be constantly lower than in the monoculture, (2) integration of G. textorii in the abalone culture changed the Vibrio compositions in the water as judged by the changes of bacteria colony types; (3) application of artificial diet led to dramatic increase of the levels in TB and TV in both systems at 12 h after application in the 24-h test and resulted in selective propagation of Vibrio in the water in the monoculture system; (4) polyculture of G. textorii with juvenile abalone in combination with feeding with live algal diet helped to maintain low levels of TV and the balance of the Vibrio composition; (5) living biomass of G. textorii was effective in preventing propagation of two purified Vibrio strains (V alginolaticus and V logei) in the water. These results provide a general basis of the dynamic changes of levels in TB and TV in a seaweed-abalone polyculture system with or without artificial diet in tanks. (c) 2005 Elsevier B.V All rights reserved.

Effects of various algal diets and starvation on larval growth and survival of Meretrix meretrix

Effects of food availability on larval growth and survival of Meretrix meretrix were studied in two experiments by feeding the larvae with different algae diets and by starving the larvae for different periods of time. Newly hatched larvae of M meretrix were fed with five different marine microalgae species, singly and in various mixtures. Best growth was with Isochrysis galbana as a single species diet. Nutritional value of the other single species diets was in the order of Dunaliella sp.> Phaeodactylum tricornutum > Platymonas subcordiformis > Pavlova viridis. Of the mixtures tested, 50% I. galbana/50% Dunaliella sp., 50% I. galbana/50% P tricornutum, and 50% 1 galbana/50% P subcordiformis, supported growth and metamorphosis equivalent to those of the I. galbana control. At 25 degrees C, larvae of M meretrix were deprived of food for various days to study the growth compensation from the outset of development. The results showed that M meretrix larvae could survive long feeding delays, and even reach metamorphosis without food added, although starvation had significant effects on growth. These results suggested that M meretrix larvae had the capacity to survive 'starvation' using alternative sources of energy. It also showed that growth, survival and metamorphosis of M meretrix were affected by many factors besides food quality and quantity. (c) 2005 Elsevier B.V. All rights reserved.

The genetic analysis and germplasm identification of the gametophytes of Undaria pinnatifida (Phaeophyceae) with RAPD method

Eleven pairs of Undaria pinnatifida (Harv.) Suringar gametophytes were identified with random amplified polymorphic DNA (RAPD) technique. After screening 100 primers, 20 ten-base primers were determined for the RAPD analysis. A total of 312 polymorphic loci were obtained, of which 97.7% were polymorphic. The primer S198 was found to distinguish all the selected Undaria pinnatifida gametophytes. The genetic distances between each two of the twenty-two U. pinnatifida gametophytes ranged from 0.080 to 0.428, while the distances to the Laminaria was 0.497 on average. After reexamination, two sequences characterized amplification region (SCAR) markers were successfully converted, which could be applied to U. pinnatifida germplasm identification. All these results demonstrated the feasibility of applying RAPD markers to germplasm characterization and identification of U. pinnatifida gametophytes, and to provide a molecular basis for Undaria breeding.

Evolutionary analysis of phycobiliproteins: Implications for their structural and functional relationships

Phycobiliproteins, together with linker polypeptides and various chromophores, are basic building blocks of phycobilisomes, a supramolecular complex with a light-harvesting function in cyanobacteria and red algae. Previous studies suggest that the different types of phycobiliproteins and the linker polypeptides originated from the same ancestor. Here we retrieve the phycobilisome-related genes from the well-annotated and even unfinished cyanobacteria genomes and find that many sites with elevated d(N)/d(S) ratios in different phycobiliprotein lineages are located in the chromophore-binding domain and the helical hairpin domains (X and Y). Covariation analyses also reveal that these sites are significantly correlated, showing strong evidence of the functional-structural importance of interactions among these residues. The potential selective pressure driving the diversification of phycobiliproteins may be related to the phycobiliprotein-chromophore microenvironment formation and the subunits interaction. Sites and genes identified here would provide targets for further research on the structural-functional role of these residues and energy transfer through the chromophores.

Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay, China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15-6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.

Annual cycle of carbon, nitrogen and phosphorus in the Bohai Sea: A model study

The annual cycle of nutrient-phytoplankton dynamics in Bohai Sea (BS) is simulated using a coupled physical-biological model in this study. By comparison, the modeled seasonal variations of nutrients and primary productivity agree with observations rather well. Although the annual cycles of chlorophyll a and primary production are both characterized by a double-peak configuration, a structural difference is still apparent: the phytoplankton biomass reaches the highest value in spring while summer is characterized by the most productivity in the BS, which can be ascribed to the combined impact of seawater temperature and zooplankton-grazing pressure on the growth of algae. Based on the validated simulations, the annual budgets of carbon, nitrogen and phosphorus are estimated, and are about 0.82 mt C surplus, 39 kt N deficit and 12kt P surplus, respectively, implying that the BS ecosystem is somewhat nitrogen limited. The contribution of two external nutrient sources, namely river discharges and resuspended sediments, to the growth of algae is also examined numerically, and it is found that the influence of river-borne nutrients mainly concentrates in estuaries, whereas the reduction of sediment-borne nutrients may significantly inhibit the onset of algae bloom in the whole BS. (c) 2007 Elsevier Ltd. All rights reserved.

Numerical investigation of nutrient limitations in the Bohai Sea

River discharges are the important freshwater and nutrient sources for Bohai Sea (BS), and have a profound impact on the local marine environment. In this paper, the annual cycles of nutrient and phytoplankton dynamics in 1980s were reproduced using a coupled biogeochemical-physical model. Based on the validated simulations, the nutrient limitation characters were further investigated by running the model with the riverine nutrient altered, first enriching nitrogen and then phosphorus. It was found that although the riverine N:P ratios in Yellow and Haihe Rivers were much higher than the Redfield number, the nitrogen enrichment was still able to enhance the algae bloom in Laizhou and Bohai Bays. On the other hand, the response of algae growth to phosphorus enrichment was not thus obvious, which suggests that the local phytoplankton dynamics was characterized by the nitrogen limitation. Simulations also show that the nitrogen enrichment is generally accompanied by the phosphorus consumption, so a shift from nitrogen limitation to phosphorus limitation may occur if such a trend continues. (C) 2010 Elsevier Ltd. All rights reserved.

The main phosphorous sources in the Changjiang estuary

Analysis using historical data on the phosphate sources in Changjiang (Yangtze River) estuary show that phosphate was supplied equally from the east, south, west and north of the estuary. These sources include the Changjiang River, the Taiwan Warm Current (TWC), a cyclone-type eddy, and the 32A degrees N Upwelling, supplying different phosphates in different times, ways and intensities. The magnitude of their supplying phosphate concentration was related with the size in the order of the Changjiang River < the TWC < the 32A degrees N Upwelling < the cyclone-type eddy, and the duration of the supplying was: the Changjiang River > the TWC > the cyclone-type eddy > the 32A degrees N Upwelling. The four sources supplied a great deal of phosphate so that the phosphate concentration in the estuary was kept above 0.2 mu mol/L in previous years, satisfying the phytoplankton growth. The horizontal and vertical distribution of the phosphate concentration showed that near shallow marine areas at 122A degrees E/31A degrees N, the TWC in low nutrient concentration became an upwelling through sea bottom and brought up nutrients from sea bottom to marine surface. In addition, horizontal distribution of phosphate concentration was consistent with that of algae: Rhizosolenia robusta, Rhizosolenia calcaravis and Skeletonema, which showed that no matter during high water or low water of Changjiang River, these species brought by the TWC became predominant species. Therefore, the authors believe that the TWC flowed from south to north along the coast and played a role in deflecting the Changjiang River flow from the southern side.

胶州湾及其邻近海域的浮游植物－种类组成与数量变化

研究胶州湾浮游植物的物种组成与时空分布的特点，对于了解该湾生态系统的现状与历史变化趋势以及生态系统对自然条件变化和人类干扰的响应具有重要意义。 本文根据2004年每月一次采集的浮游植物样品，分析了胶州湾浮游植物的物种组成和优势种的时空分布情况。调查发现浮游植物共142种，分属于6门53属，其中硅藻门40属113种，占总物种数的79.6%；甲藻门10属24种，占总物种数的16.9%；其它为金藻门1属2种，裸藻1属1种，绿藻1属2种。浮游植物丰度周年波动范围为11.12－14602.39×104cells/m3，全年平均为1857.55×104cells/m3。全年丰度最高的藻为环纹劳德藻(Lauderia annulata)，出现在2月份，而全年的丰度最高值也出现在2月份。在胶州湾中，硅藻所占比例最大，平均为97.44%，最高为99.95%，最低为82.55%。浮游植物的丰度的周年变化：有两个高峰，分别出现在2月份和10月份，是典型的温带海域双高峰的分布形式。 胶州湾浮游植物的优势种分析，采用Kikvidze等（2002）提出的确定优势种数量的计算方法，再依据各物种的优势度排序最终确定优势种。其中在多样性最低的6月和10月，优势种数目均为1种，分别为丹麦细柱藻（Leptocylindrus danicus）和中肋骨条藻（Skeletonema costatum）。而在多样性指数较低的1月、2月，优势种数目分别为2和3；而在物种多样性比较高的5月、7月、11月，由于分布相对比较分散，所以优势种数量较多。这说明这个方法可以较好地完成对胶州湾浮游植物群落分析时确定优势种的目标。优势种出现频率较多的种类为中肋骨条藻（1、3、4、5、8、10、11、12月）、洛氏角毛藻(Chaetoceros lorenzianus)（7、8、9、11、12月）、尖刺拟菱形藻(Pseudonitzschia pungens)（2、3、5、7月）、加拉星杆藻(Asteronella kariana)（1、2、3、5月）、密联角毛藻(Chaetoceros densus)（5、11、12月）、扭鞘藻(Streptothece thamesis)（7、8、9月）、夜光藻(Noctiluca scintillancs)（4、5、7月）、奇异菱形藻(Nitzschia paradoxa)（5、11、12月）,对这些种的时空分布进行了分析。 对2004年胶州湾的浮游植物数据进行分层聚类分析后，发现可以将胶州湾划分为三个海区：湾南与湾外海区、湾中西部海区、湾东海区。