Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos

With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60 min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60 min, are in the following sequence: PG > Me2SO approximate to MeOH approximate to glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG, gave the best protection at -15 degrees C. (c) 2004 Elsevier Inc. All rights reserved.

Comparative studies on the efficiency of energy transfer of two R-phycoerythrin-C-phycocyanin conjugates constructed from sodium periodate and glutaraldehyde respectively

The C-phycocyanin and the R-phycoerythrin were purified from the blue-green alga Spirulina platensis and red alga Polysiphonia urceolata respectively. Both sodium periodate and glutaraldehyde are effective coupling agents being capable of constructing the R-phycoerythrin-C-phycocyanin conjugate, which was also called phycobiliproteins energy transfer model. The two artificial conjugates constructed with different methods were purified by Sephadex G-200 chromatography respectively. Spectra analysis indicated that energy transfer occurred in the two conjugates. The conjugate with sodium periodate had the higher efficiency of energy transfer than that with glutaraldehyde conjugate.

Effect of temperature and irradiance on the growth and reproduction of Enteromorpha prolifera J. Ag. (Chlorophycophyta, Chlorophyceae)

Effect of temperature and irradiance on growth and reproduction of Enteromorpha prolifera that bloomed offshore along the Qingdao coast in summer 2008, was studied. It was showed that E. prolifera propagated mainly asexually with specific growth rate (SGR) of 10.47 at 25A degrees C/40 mu mol m(-2)s(-1). Under this condition, gametes with two flagellate formed and released in 5 days. At the beginning of the development, the unicell gamete divided into two cells with heteropolarity, and then the apical cell developed into thalli primordial cells, whereas the basal cell developed into rhizoid primordial cells. In 8-day culture, the monoplast gamete developed into juvenile germling of 240 mu m in length. Unreleased gametes can develop directly within the alga body. E. prolifera could either reproduce through lateral branching or fragmenting except apomixis revealed by Microscopic observation. On aged tissue of E. prolifera, although the degraded pigments partially remained in faded algal filaments, numerous vegetative cells could still divide actively in the algal tissues.

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.

Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae)

The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) assay, and higher than those of the positive controls in beta-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the beta-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1-F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg center dot g(-1) seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1-F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.

红毛菜发育过程及其生理基础

红毛菜（Bangia Lyngb.）属于红藻门，与紫菜属同属红毛菜科，其味道和营养都优于紫菜。目前红毛菜栽培产业已在我国福建莆田展开，但栽培技术还有待提高。海藻栽培技术的发展和成熟依赖于对其生长发育过程的认识。本研究针对红毛菜发育过程及相关光合生理展开，并初步探讨了一采自山西娘子关泉淡水红毛菜群体（FWB）的系统地位。 色素突变标记的壳孢子萌发特征表明最初两次分裂产生的4细胞决定了完整植株的形态建成。成熟植株，为雌雄异体。雌性生殖器果胞的标志性分化结构为原始受精丝，环境因子是促发原始受精丝发展的外部因素，其膨大程度随受精的延迟而增大。原孢子是主要的无性生殖孢子类型，在不良环境中，藻体也会形成内生孢子或休眠包囊，或者藻体断裂后重新形成完整的植株。 红毛菜的生长发育很大程度上受环境因子的控制。高温不利于配子体的发育，15-20 ºC比较适宜。红毛菜无性繁殖的最适温度-光照组合为20 ºC-8 h，有性繁殖为15 ºC-12h。 不同发育阶段，PSII实际光合效率（Y(II)）与细胞的健康状况以及光合器官完整性及其在细胞内的分布有关，而与细胞的类型关系不大。健康的假根细胞、已分化未成熟的精子以及果孢子细胞均具有很高的Y(II)。色素体由中间位变为围周位，中央大液泡（营养藻丝）和大小纤维囊泡（成熟孢子与精子）的产生，使得细胞Y(II)降低。刚放散的壳孢子Y(II)很低，说明在壳孢子由贝壳基质释放到自由水体过程，光合作用受到一定程度抑制；而2h后，Y(II)开始恢复，rbcL的转录水平非常高，为孢子的萌发储备物质和能量需求。 在失水和低盐胁迫下，藻体均维持较高的Y(II)。干出处理至藻体重量不再变化，复水后Y(II)可回复初始水平。海生红毛菜在100％淡水培养基中（约20ºC）培养7天后，部分雄性藻体依然活着。从而体现了红毛菜位居高潮带的生理优势。 FWB终生行无性繁殖，藻体形态与发生以及染色体数目（4条）与海生群体没有区别。而rbcL-rbcS Spacer序列显示，红毛菜海生群体（无性和有性）具有完全相同的序列，而FWB与它们有5bp差异，但是与欧洲、北美地区的淡水群体仅1bp不同，初步说明所有淡水红毛菜群体具有共同的原始起源。

Feeding level-scaled retention efficiency, growth and energy partitioning of a marine detritivorous fish, redlip mullet (Liza haematocheila T. & S.)

The effects of feeding level on growth, retention efficiency, faeces production and energy partitioning of redlip mullet were studied. A practical diet was used and fed at six levels from starvation, 1%, 2%, 3%, 4% of body weight (BW) to satiation for 3 weeks. The temperature was kept at 24 +/- 1 degrees C. Reducing the feeding amount resulted in significantly lower weight gain, and retention efficiency was significantly affected by feeding levels and attained the maximum at maximum feeding intake. Feeding 2% BW was the minimum required for fish to maintain growth. Fish carcass composition under different feeding levels could be divided into three groups: (1) starvation and FL1; (2) FL2 and FL3 and (3) FL4 and satiation, with significant differences among the groups but no differences in the groups except that ash content remained at constant value. Body composition of fish of group 2 was close to initial fish. The thermal-unit coefficient was 0.0381 at satiation, and significantly increased with increasing feeding levels. In order to accurately estimate basal metabolism (HeE), another trial on the relationship between HeE (kJ) and BW (g) was carried out. An exponential curve as HeE=0.1255BW(0.8386) explained this relationship. Intake energy (IE) increased from 11.30 to 63.08 kJ per fish, matching with different feeding levels. Energy allocated to growth of IE decreased with reducing feeding amount. There was a linear relationship between metabolism energy and retention energy in percentage.

Effects of single and mixed diatom diets on the reproduction of copepod Calanus sinicus

Under laboratory conditions, the potential influence of diatom diets on reproduction of zoo-plankton Calanus sinicus was studied. Four diatom diet ingredients: Skeletonema costatum (SC), Chaetoceros muelleri (CM), Phaeodactylum tricornutum (PT), diatom mixture (MIX) and a control diet: the flagellate Platymonas subordiformis (PS), were used at the same carbon concentrations of 2.0 mu g mL(-1) C. In a period of 17-day laboratory experiment, the effects of these algae diets on egg production and hatching success of the copepod Calanus sinicus were examined. The diets were analyzed for fatty acid content as an indicator of food quality. The results showed that the female survival of all treatments reached more than 80% except PT. Comparing to the initial value, egg production of Calanus sinicus was reduced in diatom diets (PT, CM), but remained in normal level in SC and MIX, indicating that some single diatom diets had a negative effect on the egg production of Calanus sinicus. Feeding with mixed food however can eliminate this negative effect. Among all the treatments, hatching success in filtered seawater was significantly higher than in algal exudates, indicating that not only diatoms but also other phytoplankton in certain concentration can release extracelluar substance that may inhibit eggs from hatching. Fatty acid analysis showed that both egg production rate and hatching success were negatively correlated to the ratio of 20:5 omega 3 and 14:0 in fatty acid composition.

Summer reproduction of the planktonic copepod Calanus sinicus in the Yellow Sea: influences of high surface temperature and cold bottom water

We have observed that Calanus sinicus retreated from neritic areas in the Yellow Sea and concentrated in the Yellow Sea Cold Bottom Water (YSCBW) area in summer. To investigate the summer reproductive strategy of C. sinicus in this situation, effects of high temperature on reproduction and hatching, as well as geographical variation of in situ egg production rate, were studied by onboard incubation in August 2001. Diel vertical migration (DVM) of females was investigated within and outside the YSCBW, respectively. Onboard incubation at 27 degrees C (i.e. surface temperature) resulted in lower fecundities than that at 9.8 and 12 degrees C (i.e. bottom temperature inside and outside the YSCBW) together with decreased hatching rates and increased naupliar malformation. Egg production was more active at stations outside the YSCBW than inside, where chlorophyll-a concentration was also relatively low. Females inside the YSCBW underwent DVM although they rarely entered the surface layer, but DVM was not observed outside the YSCBW. We conclude that surface temperature in summer has deleterious effects on C. sinicus egg production and hatching, and that it cannot reproduce successfully over the whole area. Inside the YSCBW, egg production is depressed by low food availability, while females outside suffer from high temperatures because of strong vertical mixing.

Seasonal variation of reproduction rates and body size of Calanus sinicus in the Southern Yellow Sea, China

Annual variations of egg production rate (EPR) and clutch size of Calanus sinicus, as well as body size of females (prosome length and dry weight), were investigated at a series of stations in the Southern Yellow Sea by onboard incubation. Calanus sinicus was spawning in all the 11 cruises investigated, and the annual variation of EPR was bimodal. Monthly average EPR was highest from May to July, respectively, 5.97, 5.36 and 6.30 eggs female(-1) d(-1), then decreased dramatically to only 1.37 eggs female(-1) d(-1) in August and attained the lowest 1.07 eggs female(-1) d(-1) in October. In November, average EPR increased again to 4.31 eggs female(-1) d(-1). Seasonal variation of clutch size was similar to EPR, except that it decreased gradually after August rather than dramatically as did EPR. Prosome length of females was maximum in May and minimum in October, but dry weight was highest in November. Monthly average EPR correlated better with prosome length than dry weight, while clutch size was rather determined by dry weight of females. It is suggested that egg production of C. sinicus was active during two discontinuous periods when both surface and bottom temperature fell into its favorite range (i.e. 10-23degreesC), and different reproductive strategies were adopted in these two reproductive peaks: other than the highest EPR, longer prosome length was also achieved by C. sinicus from May to July, while females in November developed shorter bodies but accumulated more energy for reproduction.

Spring and autumn reproduction of Calanus sinicus in the Yellow Sea

The fecundity, hatching success and naupliar survival of Calanus sinicus were studied in the Yellow Sea during research cruises in April and October 2006, with emphasis on the regulation of reproduction. During both cruises, the egg production rate (EPR) showed large spatial variations (0 to 25.4 eggs female(-1) d(-1)), generally coinciding with the food availability. In April, the abundant phytoplankton and ciliates in the study area supported active reproduction, which would probably initiate the annual population development. In October, females remained immature in the Yellow Sea Cold Water Mass, likely due to the unfavorable environment (poor food and low temperature). However, reproduction and recruitment were high in the neritic region, which may explain the local population recovery in late autumn. Hatching success varied markedly among stations in April (4 to 85%), whereas it was high overall in the neritic region during October (> 90%). Based on the potential recruitment rates, the spring recruitment would be more important for the annual population dynamics, Female gonad maturity, body size and lipid reserve were examined in relation to fecundity. Regression analyses suggested that the reproductive index (defined as the proportion of females with mature gonads) could be an indicator for the EPR of C. sinicus. Among the regulating factors, external food (ciliates and phytoplankton) seems essential for reproduction, whereas inner lipid reserve may mainly serve metabolic needs. Moreover, fecundity is positively related to body size but independent of temperature, which might exert indirect influences on reproduction.

Effects of diatoms on reproduction and growth of marine copepods

Two hot spots in marine ecology, deleterious effects of diatoms and feeding selectivity of copepods, as well as new progress on these two issues achieved in the recent ten years, are reviewed. These two issues are considered correlated closely. Diatoms and their metabolites can induce deleterious effects on growth, reproduction and development of copepods, including increase of mortality and decrease of egg production, hatching and growth rates. Such negative effects, resulting from either chemical toxin or nutritional deficiency, can be conquered in natural environments by diverse feeding. It is therefore concluded that deleterious effects of diatoms observed in laboratory or during blooming period are only a special case that accommodation of feeding strategy of copepods is disabled. To understand their feeding strategy in natural environments is a prerequisite to explaining the mechanisms of deleterious effects caused by diatoms, and makes it possible to re-evaluate the energy flow in marine ecosystems.

Effects of different diets on the reproduction and naupliar development of the copepod Acartia bifilosa

The influence of diatoms on the reproduction and naupliar development of Acartia bifilosa was investigated under laboratory conditions, comparing initial in situ values and laboratory-food treatments. Egg production by A. bifilosa was significantly reduced by one diatom diet (Phaeodactylum tricornutum: Pt) and by two non-diatom diets (Platymonas subordiformis: Ps and Nannochloropsis oculata: No). It was less affected by the other diatom diet (Skeletonema costatum: Sc) or by two mixed-food treatments (D-mix and DG-mix), composed of two diatoms (Pt, Sc) and four species (Pt, Sc, Ps and No), respectively. The negative effect of Pt was eliminated when adult copepods were offered mixed-food diets. There were no significant differences between the hatching success values observed in filtered seawater and in algal exudates, indicating that diatoms did not produce active dissolved toxic substances under the different food concentrations tested. The mortality rate of nauplii was higher with Pt than the other diets, suggesting that this diatom species had a negative effect on egg production, hatching success and naupliar survival simultaneously. Compared to other diets, No and Pt were not beneficial food sources for reproduction and for female and larval survival. (c) 2007 Elsevier B.V. All rights reserved.