CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 muL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst. (C) 2000 by Elsevier Science Inc.

Daily timing of emersion and elevated atmospheric CO2 concentration affect photosynthetic performance of the intertidal macroalga Ulva lactuca (Chlorophyta) in sunlight

The lunar day differs in length from the solar day so that times of low tide vary from day to day. Thus, aerial exposure of intertidal seaweeds may be during the day or during the night. We measured photosynthetic CO, assimilation rates of the intertidal green macroalga Ulva lactuca during exposures of varied daily timings during sunny days of summer to establish how photosynthetic performance responds to emersion timing under varied CO2 levels [at ambient (360 ppmv) and 2x ambient (720 ppmv) atmospheric CO2 concentrations]. There was an increase in net photosynthetic rates following some duration of exposure when the initial timing of exposure occurred during early morning (06.30 h) and late afternoon (17.15 h). In contrast, net rates exhibited a sharp decline with exposure duration when the initial timing of exposure occurred at 09.30 h, 15.30 h and especially at noon (12.30 h), implying the occurrence of a severe photoinhibition resulting from mid-day insolation. Doubled atmospheric CO2 concentration significantly enhanced the emersed photosynthetic rates, indicating that the emersed photosynthesis is CO2-limited at ambient CO2 levels. However, increasing CO2 barely stimulates the emersed photosynthetic rates during mid-day insolation.

Responses of dark respiration in the light to desiccation and temperature in the intertidal macroalga, Ulva lactuca (Chorophyta) during emersion

Dark respiration (nonphotorespiratory mitochondrial CO2 release) in the light (R-L) of the intertidal macroalga Ulva lactuca (Chorophyta) during emersion was investigated with respect to its response to variations in temperature and desiccation. R-L was estimated by CO2 gas-exchange analysis using the Kok effect method, whereas dark respiration in darkness (R-D) was determined from CO2 release at zero light. Rates of R, were significantly and consistently lower than those of R-D in emersed U. lactuca across all the temperature and desiccation levels measured. This demonstrated that dark respiration was partially depressed in the light, with the percentage inhibition ranging from 32 to 62%. Desiccation exerted a negative effect on R-L and R-D at a high temperature, 33 degrees C, whereas it had much less effect on respiration at low and moderate temperatures, 23 and 28 degrees C. In general, R-L and R-D increased with increasing temperature in U. lactuca during all stages of emersion but responded less positively to temperature change with increasing desiccation. Additionally, the Q(10) value (i.e. the proportional increase of respiration for each 10 degrees C rise in temperature) for R-L calculated over the temperature range of 23 to 33 degrees C was significantly higher than that for R-D in U. lactuca during the initial stages of emersion. Respiratory carbon loss as a percentage of gross photosynthetic carbon gain increased with increasing temperature and/or desiccation but was significantly reduced when estimated using R-L rather than R-D. It is suggested that measurements of R-L and how it changes in a variable environment are as important as estimates of R-D and photosynthesis in determining simultaneous balance between photosynthetic carbon uptake and respiratory carbon loss and in modeling the net daily carbon gain for an intertidal macroalga.

Photosynthetic recovery of desiccated intertidal seaweeds after rehydration

Intertidal seaweeds experience periodical desiccation and rehydration to different extents due to the tidal cycles and their vertical distributions. Their photosynthetic recovery process during the rehydration may show different patterns among the seaweeds from different zonations or depths at intertidal zone. In this study 12 species of seaweeds collected from the upper, middle, lower and sublittoral zones were examined. The relationship of the photosynthetic recovery to vertical distribution was assessed by comparing their patterns of photosynthetic and respiratory performances after rehydration following desiccation. Both the photosynthesis and dark respiration declined during emersion, showing certain degrees of recovery after re-immersion into seawater for most species, but the extents were markedly different from one species to the other. The species from upper intertidal zone after being rehydrated for 1 hour, following 2 hours of desiccation, achieved 100 % recovery of their initial physiological activity, while most of the lower or sublittoral species did not achieve full recovery. It is the ability to withstand desiccation stress (fast recovery during rehydration), but not that to avoid desiccation (water retaining ability) that determines the distribution of intertidal seaweeds. Such physiological behavior during rehydration after desiccation reflects the adaptive strategy of intertidal seaweeds against desiccation and their capability of primary production in the process of rehydration.

Ecophysiological characteristics of four intertidal marine macroalgae during emersion along Shantou coast of China, with a special reference to the relationship of photosynthesis and CO2

Intertidal marine macroalgae experience periodical exposures during low tide due to their zonational distribution. The duration of such emersion leads to different exposures of the plants to light and aerial CO2, which then affect the physiology of them to different extents. The ecophysiological responses to light and CO2 were investigated during emersion in two red algae Gloiopeltis furcata and Gigartina intermedia, and two brown algae Petalonia fascia and Sargassum hemiphyllum, growing along the Shantou coast of China. The light-saturated net photosynthesis in G. furcata and P. fascia showed an increase followed by slightly desiccation, whereas that in G. intermedia and S. hemiphyllum exhibited a continuous decrease with water loss. In addition, the upper-zonated G. furcata and P. fascia, exhibited higher photosynthetic tolerance to desiccation and required higher light level to saturate their photosynthesis than the lower-zonated G. intemedia and S. hemiphyllum. Desiccation had less effect on dark respiration in these four algae compared with photosynthesis. The light-saturated net photosynthesis increased with increased CO2 concentrations, being saturated at CO2 concentrations higher than the present atmospheric level in G. furcata, G. intermedia and S. hemiphyllum during emersion. It was evident that the relative enhancement of photosynthesis by elevated CO, in those three algae increased, though the absolute values of photosynthetic enhancement owing to CO2 increase were reduced when the desiccation statuses became more severe. However, in the case of desiccated P. fascia (water loss being greater than 20 %), light saturated net photosynthesis was saturated with current ambient atmospheric CO2 level. It is proposed that increasing atmospheric CO2 will enhance the daily photosynthetic production in intertidal macroalgae by varied extents that were related to the species and zonation.

Cultivation of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura: mass production of zygote-derived seedlings under commercial cultivation conditions, a case study experience

Mariculture of the brown alga Hizikia fusiformis (Harvey) Okamura as an export-oriented human food has been there more for than 20 years in China. It is now one of the five major farmed algal species along the Chinese coast. Stable and sufficient supply of young seedlings for scaling up the cultivation has been a problem throughout the farming history of this species due to the unique dioecious life cycle and relatively short time window of sexual reproduction in nature. These two factors led to a practical difficulty in obtaining zygotes at identical developmental stage in viable amounts for seedling production. A key solution to this problem is to control the synchronization of the receptacle development and to realize the simultaneous discharge of male and female gametes, such that the fertilization rate could be greatly enhanced. Focusing on one of the farmed populations in this report, we present our results on mass production of seedlings using the synchronization technique on a large scale performed in 2007. Totally 5.5 hundred million embryos were obtained from 100 kg female sporophytes. The seedlings were raised up to 3.5 mm in length in greenhouse tanks over a month and were further grown in open sea for over 3 months at two experimental sites. The success of mass production of seedlings in this alga helped to lay the basis for future trials in other species in the genus of Sargassum that have identical life cycle.

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 +/- 0.07 mol H-2/mol glucose (mean +/- S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 +/- 0.03 mol H-2/mol glucose, 0.17 +/- 0.01 mol H-2/mol glucose, 0.11 +/- 0.01 mol H-2/mol glucose and 0.20 +/- 0.04 mol H-2/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp., However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Evidences of the intertidal red alga Grateloupia turuturu in turning Vibrio parahaemolyticus into non-culturable state in the presence of light

Grateloupia turuturu, previously known as Grateloupia doryphora, has been widely reported to be an invasive algal species. There are no studies to relate the impact of its existence on its surrounding environment. In this paper, we present our results to show that about 70% of individuals collected from the field could turn Vibrio parahaemolyticus into non-culturable state on both selective (TCBS) and non-selective (2216E) culture medium in 24 h in the presence of light in live algal culture. Total bacteria counts on TCBS and 2216E plates dropped from the initial 565 (174) and 1192 (60) cfu ml(-1) respectively to zero in 24 h. This effect disappeared when the alga was grown in darkness. The same effect was not found in two other intertidal macroalgae Laminaria japonica and Palmaria palmata. Further tests showed that the settlement ability of bacteria in seawater was impaired significantly in the presence of this alga in comparison with three other algal species. (c) 2006 Elsevier B.V. All rights reserved.

Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea

Magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats. Because of their fastidious requirements for growth conditions, only very few axenic MTB cultures have been obtained worldwide. In this study, we report a novel marine magnetotactic spirillum axenic culture, designated as QH-2, isolated from the China Sea. It was able to grow in semi-solid or liquid chemically defined medium. The cells were amphitrichously flagellated and contained one single magnetosome chain with an average number of 16 magnetosomes per cell. Phosphate and lipid granules were also observed in the cells. Both rock magnetism and energy-dispersive X-ray spectroscopy characterizations indicated that the magnetosomes in QH-2 were single-domain magnetites (Fe3O4). QH-2 cells swam mostly in a straight line at a velocity of 20-50 mu m/s and occasionally changed to a helical motion. Unlike other magnetotactic spirilla. QH-2 cells responded to light illumination. As a consequence of illumination, the cells changed the direction in which they swam from parallel to the magnetic field to antiparallel. This response appears to be similar to the effect of an increase in [O-2]. Analysis of the QH-2 16S rRNA sequence showed that it had greater than 11% sequence divergence from freshwater magnetotactic spirilla. Thus, the marine QH-2 strain seems to be both phylogenetically and magnetotactically distinct from the freshwater Magnetospirillum spp. studied previously. (C) 2010 Elsevier Masson SAS. All rights reserved.

Summertime community structure of intertidal macrobenthos in Changdao Archipelago, Shandong Province, China

The community structure of intertidal macrobenthos in Changdao Archipelago (north of Shandong Peninsula, between Bohai Bay and the northern Yellow Sea) was examined based on samples collected from 14 stations in five transects in June 2007. Three stations corresponding to high, medium and low tidal areas were set up for each transect. A total of 68 macrobenthic species were found in the research region, most of which belonged to Mollusca and Crustacea. The average abundance and biomass of the macrobenthos was 1383 ind./m(2) and 372.41 g/m(2), respectively. The use of an arbitrary similarity level of 20% resulted in identification of five groups among the 14 stations in the research region. There were remarkable differences in the biomass, abundance and Shannon-Wiener diversity index of the different sediments. Specifically, the order of biomass was rocky shores > gravel > mud-sand > coarse sand > stiff mud, while the order of abundance was rocky shores > coarse sand > mud-sand > gravel > stiff mud, and that of the diversity index was mud-sand > gravel > stiff mud > rocky shores > coarse sand. The above results revealed that the sediment type was the most important factor affecting the structure of the macrobenthic community of the intertidal zone.

Holosticha hamulata n. sp and Holosticha heterofoissneri Hu and Song, 2001, two urostylid ciliates (Protozoa, Ciliophora) from intertidal sediments of the Yellow Sea

Two marine urostylid ciliates, Holosticha hamulata n. sp. and Holosticha heterofoissneri Hu and Song, 2001, were investigated using live observation and protargol impregnation. Both species were isolated from Korean intertidal sediments of the Yellow Sea. Holosticha hamulata measures about 150 x 25 pro in vivo, and is characterized by a tripartite body shape with a narrow head, an inflated trunk, and a tail that distally projects ventrally forming a hook-like structure. It is the characteristic body shape that distinguishes H. hamulata distinctly from congeners. Holosticha hamulata differs from H. heterofoissneri, possibly the nearest relative, also by the location of the contractile vacuole (ahead of mid-body versus near posterior body third) and the configuration of the macronucleus (on average, 33 scattered nodules assuming a Y-shape versus 17 nodules that may form a U shape). The average number of the macronuclear nodules is a pronounced feature showing great consistency in populations of each species. However, their arrangement is variable in H. heterofoissneri where the nodules are basically scattered or connected by fine fibers forming an elongate U-shape. The location of the contractile vacuole as a taxonomic feature is discussed and a dichotomous key to the species of Holosticha sensu stricto is provided.