Effect of disinfectants on the hatching of marine rotifer resting eggs Brachionus plicatilis Müller

Scanning electron microscopy indicated that stored resting eggs of the marine rotifer Brachionus plicatilis Müller were colonized by numerous bacterial cells that affect the hatchability of resting eggs. The effectiveness of chemical disinfectants on the hatching of the resting eggs was studied using selected chemicals and antibacterial drags. The hatching success rate of the fresh and stored eggs was 26 ± 5.8% and 0%, respectively. Sodium hypochlorite (1.0 p.p.m.) increased the hatching rate to 48 ± 1.3% in freshly-collected and 28 ± 1.8% in stored eggs. Physical disintegration of the bacterial mat occurred after 1 h of treatment with sodium hypochlorite. Among antibacterial drugs, sodium nifurstyrenate (Erubaju) enhanced the hatching success rate to 40 ± 1.2% and 20 ± 1.1% for the freshly-collected eggs and stored resting eggs, respectively. However, tetracycline and oxytetracycline did not significantly influence the hatching of the stored eggs. These observations are discussed in relation to the development of an improved method for the processing and hatching of rotifer resting eggs.     

Use of a potential probiotic Lactococcus lactis AR21 strain for the enhancement of growth in the rotifer Brachionus plicatilis (Müller)

The effect of a potential probiotic on the growth performance of a rotifer and its inhibition against Vibrio anguillarum was studied. Probiotic strain AR21 had no significant observable effect on the growth rate of rotifers under optimal culture conditions in three consecutive experiments. In the first and second experiments, the AR21 strain exhibited an inhibitory effect against the V, anguillarum strain when rotifer cultures were maintained at a suboptimal feeding regime. The growth rate of the rotifers in suboptimal feeding conditions was significantly higher in the treatment receiving AR21 and V. anguillarum than in the treatment where only V. anguillarum was added.     

Essential fatty acid enrichment of cultured rotifers (Brachionus plicatilis, Müller) using frozen-concentrated microalgae

There is a growing interest in preserving microalgal preparations to maintain constant properties over a long period. The aim is to ensure sufficient delivery of essential fatty acids (and other key nutrients) to mollusc and crustacean larvae and to zooplankton used as live prey in the first feeding of fish larvae. For example, the rotifer Brachionus plicatilis has to be enriched with polyunsaturated fatty acids (PUFA) prior to fish feeding. We used four microalgal species [ Isochrysis galbana (T-ISO), Chaetoceros muelleri (CHGRA), Pavlova lutheri (MONO), and Nannochloropsis sp.] both as fresh culture or in a frozen-concentrated form to enrich rotifers. Overall, rotifers had similar relative fatty acid levels when fed the frozen-concentrated or fresh microalgal diets. The levels of 20:4n-6, 22:6n-3, and 20:5n-3 between B. plicatilis and the microalgal diets were linearly correlated. The fatty acid 20:4n-6 was the most readily assimilated: the content found in rotifers reached half the level measured in the microalgal diets. Our results indicate that both the fresh and frozen-concentrated forms of the four microalgal species can be used to enrich PUFA levels in rotifers. Further experiments should be conducted to test if assimilation differs when rotifers are enriched with mono- or multispecific microalgal preparations.     

不同脂肪源对褶皱臂尾轮虫脂类和脂肪酸组成的影

以褶皱臂尾轮虫（Brachionus plicatilis)为实验动物并设计不同脂肪源饵料，分别为：面包酵母Saccharomyces cerevisice（对照组），微绿球藻Nannochlorpisis oculata(A)，5%大豆磷脂 5%鱼油 90%面包酵母（C）。结果表明：（1）接受不同脂肪源的轮虫的脂肪酸组成显著不同，尤其是高度不饱和脂肪酸（HUFA，20C）含量存在显著差异，说明饵料中HUFA含量对轮虫体内相应脂肪酸的含量有显著影响。投喂饵料C使轮虫的脂类HUFA水平得到了强化，提高了轮虫的营养价值。（2）轮虫脂类的HUFA水平不仅由饵料中脂类的相应脂肪酸组成决定，而且轮虫脂类HUFA的提高和强化效果与饵料中HUFA的化学形态密切相关。甘油三酯型饵料HUFA的强化轮虫效果高于磷脂型的HUFA。（3）将富含HUFA的鱼油直接添加在干性饵料如面包酵母中，而不是通过对鲁油乳化后直接强化轮虫，同样能取得良好的强化效果，且操作简便、不易污染水质，适用于轮虫的规模生产。     

Effects of selenium supplementation on the antioxidation response in the rotifer (Brachionus plicatilis)

In the present study, the effects of selenium (Se) enrichment on the rotifer, Brachionus plicatilis, were investigated. Based on the results of six Se treatments obtained from triplicate experiments, we found that the efficiency of bioaccumulation by Se from non‐acid‐soluble sources was 62‐fold greater than that from acid‐soluble sources (Se‐Chlorella 15.6 ± 1.81% vs. selenite 0.25 ± 0.03%). However, supplementation of both acid‐soluble Se at low concentrations (≤40 µg per thousand rotifers) and Se‐Chlorella enhanced rotifer lifespan, increased the rate of spawning, inhibited lipid peroxidation, decreased levels of intracellular oxidization products and maintained the stability of mitochondrial membranes. These positive effects were correlated with the activities of antioxidant enzymes (e.g. catalase‐ and guaiacol‐dependent peroxidase) which increased along with the low Se concentrations (<80 µg per thousand rotifers). In contrast, the opposite effects were found when the rotifers were exposed to high Se concentrations (≥80 µg per thousand rotifers). The antioxidation and toxicity effects appeared to be correlated with the levels of total Se bioaccumulation. Above all, selenite, which is more toxic than Se‐chlorella, is less readily accumulated and biologically active. Se‐enriched rotifers fed by Se‐Chlorella are a fish larvae food supplement in aquaculture.     

轮虫培养过程中生长及环境变化特征分析

通过1000ml烧杯、5L全自动生物反应器、常规的大池（5m^3）以及加处臭氧和生物过滤装置的大池（约5m^3）这四种培养系统,对在轮虫培养过程中轮虫的生长特征,以及pH、溶氧、氨氮及亚硝态氮等水质参数变化特征进行了较系统的探讨,研究结果可为轮虫密度培养工艺及培养系统的优化奠定基础。     

Insulin/insulin-like growth factor-like activity in the aqueous extracts of the rotifer Brachionus plicatilis

Insulin/insulin-like growth factor (IGF)-like signaling plays important roles in the aging processes of various animals. However, little is known about this signaling in rotifers, which have been used as a model animal in aging studies. Here we report that the aqueous extracts of the rotifer Brachionus plicatilis show activity similar to that of insulin/IGFs. Rotifers were cultured under four different feeding regimens (fed, starved for about ten days, or re-fed for 30 and 120 min after starvation), and then their aqueous extracts were added to culture media of rat L6 myoblasts. Treatment with these extracts increased the phosphorylation levels of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and two Akt substrates of approximately 48 and 60 kDa, and these phosphorylations were diminished when cells were preincubated with specific inhibitors of their upstream kinases (MAPK/ERK kinase and phosphoinositide 3-kinase, respectively). Furthermore, the extracts from fed rotifers provoked higher phosphorylation levels of MAPK/ERK and Akt substrates than the extract from starved rotifers, suggesting that the production of substance(s) with insulin/IGF-like activity is stimulated upon feeding in the rotifer.     

Selectivity of the rotifer Brachionus plicatilis fed mixtures of algal species with various cell volumes and cell densities

The ability for food selectivity of rotifer Brachionus plicatiliswas studied in the laboratory by feeding mixtures of microalgae with various cell volumes. Chlorella sp. (≈22 µm³) was the reference algal species, and Asteromonas gracilis, (Chlorophyta) (≈2150 µm³), Tetraselmis suesica (≈268 µm³), Dunaliella salina (≈52 µm³) and Chaetoceros sp. (≈150 µm³) the experimental species. Each was mixed with Chlorella and fed in three experiments. In the first experiment, filtration and ingestion rates of rotifers each fed with algae revealed that the highest values were measured with the mixture of Chlorella + Asteromonas, and the lowest for Chlorella + Chaetoceros. In the second and the third experiments, by using several combinations of algal densities with the mixture of Chlorella + Asteromonas, a selectivity ability of the rotifers for Asteromonas was found. A hypothesis is presented that accounts for the preference of rotifers for Asteromonas, which is suggested as a new candidate species for use in live food production of fish hatcheries.     

Growth, filtration and ingestion rate of the rotifer Brachionus plicatilis fed with large (Asteromonas gracilis) and small (Chlorella sp.) celled algal species

The rotifer Brachionus plicatilis was fed in experimental conditions with a small celled (2–5 μm) Chlorella sp. and a large celled (16–22 μm) Asteromonas gracilis algae. The specific growth rate (SGR) of rotifers fed Asteromonas (maximum 0.79) was statistically higher than that for rotifers fed Chlorella (maximium 0.61). The filtration and ingestion rates using different rotifer and algal densities exhibited certain maxima depending on the species, the cell density and the condition of the rotifers. The filtration rate was higher with Asteromonas and, although ingestion rate was lower than with Chlorella, the ingestion in terms of cell volume was 10‐fold higher. It seems that B. plicatilis ingests the larger cell diameter algal species more efficiently than the smaller species that is usually used for its mass culture.     

不同饵料对褶皱臂尾轮虫种群生长繁殖的影响↑（*）

用３种饵料、４个组合培养褶皱臂尾轮虫,研究饵料对轮虫种群生长、繁殖的影响。结果表明：用光合细菌和鲜酵母混合为饵组,轮虫的最高培养密度达到了１８２０ｍＬ－１,高于其它３组;轮虫的日平均增殖率为０．４３５１,比光合细菌组和鲜酵母组分别增加了６．４％和２５．０％。对轮虫生长、繁殖有明显的促进作用。     

Elimination of the associated microbial community and bioencapsulation of bacteria in the rotifer Brachionus plicatilis

The bioencapsulation of live bacteria in the rotifer Brachionus plicatilis was determined under monoxenic conditions. The first objective was to evaluate the microbiota of the rotifer during intensive production and to obtain sterile rotifer cultures starting from adult females or amictic eggs using PVP-Iodine, Hydrogen peroxide or antibiotic mixtures. In the rotifers, the proportion of vibrios increased significantly during the mass production, displacing other unidentified marine bacteria. Rotifers, in the absence of culturable bacteria were obtained starting from amictic eggs and using Trimetroprim-sulfametoxasole (Bactrim Roche®) at 10 ml l^–1. The effect of members of Vibrionaceae on the survival and growth rate of rotifers was determined under monoxenic conditions. The survival of rotifers was not affected in the presence of different isolates, while amictic egg formation occurred and the populations increased when the strains Vibrio proteolyticus C279 and Aeromonas media C226 were tested. All isolates were successfully incorporated in the rotifers, since there was no significant difference between the numbers of bioencapsulated cells of different strains of isolates. The results show that it is possible to replace the microbial community in rotifer cultures, started from disinfected amictic eggs, with selected bacterial strains. This could be used as a tool for future studies to reveal the role of specific bacteria on first larval stages of marine fish species.     

Diluted rotifer, Brachionus plicatilis (Muller), extract as culture medium stimulates hatching of red sea bream, Pagrus major (Temminck & Schlegel)

Hatching rates and hatching times (50% hatch) of red sea bream, Pagrus major (Temminck & Schlegel), eggs in culture media that were prepared to dilute rotifer Brachionus plicatilis Muller extract at 4000-, 6000-. 8000- and 10 000-fold with artificial sea water (σ₁₅, = 24.0) were investigated. Although differences in hatching rate between the culture media and control artificial sea water were negligible, hatching time was significantly shorter in media at 4000- and 6000-fold dilutions than in media at 8000- and 10 000-fold dilutions and in the control artificial sea water, when the eggs immediately post spawning were incubated. The hatching rates of eggs at three developmental stages, immediately post-spawning, gastrula stage and heart-beat initiation stage, incubated respectively in the medium at 6000-fold dilution and the control, did not differ either, yet the hatching times at each egg stage in the medium were significantly shorter than that in the control. These results suggest that hatching enzyme secretion from the hatching glands is promoted to perceive chemical stimuli in the water-soluble fraction of rotifer extract via the embryo's chemoreceptors, from the heart-beat initiation stage to immediately before hatching, thus shortening the hatching time.     

Semi‐continuous mass culture of rotifers (Brachionus plicatilis) using an automatic feeder

This study describes the rotifer mass culture protocol developed at the Institute of Aquaculture (HCMR) in Crete. Rotifers were cultured semi‐continuously at a density of 257.6 ± 2.6 ind. mL^?1, with a daily dilution rate of 25%. An automatic feeder was used to administer dry feed (Selco Sparkle^®) every 10 min. A number of factors were investigated so as to provide an in‐depth analysis of the advantages and disadvantages of the two systems, especially when compared with traditional protocols such as batch culture. The use of these two systems resulted in a culture of considerable duration (41 days) and daily yield (0.21 ± 0.01 ind. 10⁶ day^?1 L^?1), using exclusively dry feed. Both systems can be easily applied in hatcheries, requiring few modifications on existing protocols and contribute to a stable and predictable production. Further improvements include manipulation of the dilution rate, food ration, culture volume and duration, as well as choice of a phytoplankton‐based diet.     

Effects of feeding regime and probionts on the diverting microbial communities in rotifer Brachionus culture

Rotifer growth performance and microbial community changes associated with rotifer cultures were monitored while different feed types (Nannochloropsis oculata paste and the commercial yeast based feed CS-3000), different regimes (daily changes, changes per batch and no changes) and mixtures of three probionts (Phenylobacterium sp.; Gluconobacter sp. and Paracoccus denitrificans) were provided. It was shown that the dominant bacterial species in the cultures receiving either N. oculata or CS-3000 were different. However, in cultures receiving both feeds (either switching between feeds on a daily basis or on a batch basis), a high similarity in microbial community fingerprint was found. The presence of probionts was detected by the end of four batch culture cycles in spite of strong shifts of the bacterial community. By group discriminant analysis, it was found that Phenylobacterium sp. and Paracoccus sp. contributed positively to the CS-3000-fed group, while Gluconobacter sp. contributed positively to the N. oculata-fed group, although they did not appear as very dominant species.     

维生素B12对圆型臂尾轮虫种群增长、繁殖和个体大小的影响

为了解维生素B 12 对海水圆型臂尾轮虫( Brachionus rotundiformis )种群增长、繁殖和个体大小的影响,采用四种不同浓度的维生素B 12 (0、0.3、0.6、1.2 μg/mL)强化培养轮虫7 d。每天测定轮虫的种群密度和怀卵率,每隔一天测定轮虫背甲长和宽。试验结果显示:0.3 μg/mL维生素B 12 组轮虫的种群增长、怀卵率显著高于其他组( P<0.05);0.3 μg/mL维生素B 12 组轮虫的背甲长和宽均显著小于其他三组( P>0.05),而其他三组差异不显著。试验结论:在本试验条件下,维生素B 12 对圆型臂尾轮虫种群增长、繁殖和个体大小影响的最适浓度为0.3 μg/mL。     

Comparison of rotifer culture quality with yeast plus oil and algal-based cultivation diets

The culture quality of rotifers, Brachionus plicatilis, was compared for yeast and algal-based cultivation diets. The rotifer quality was evaluated based on factors which may affect survival and growth of marine fish larvae, including individual length and biomass, nutritional value, bacterial content, and rotifer viability. The diets used were monocultures of Tetraselmis sp., Isochrysis galbana, Pavlova lutheri, and baker's yeast plus capelin oil.Rotifers grown to early stationary phase with equal food rations exhibited equal length distribution, but the rotifer individual biomass was 10–25% higher for yeast-grown rotifers than for algal-grown rotifers.All diets gave equal protein content of individual rotifers. The lipid content was slightly higher and the ratio of protein to lipid was slightly lower with yeast plus capelin oil than with algae. The fatty acid composition of the rotifers was closely related to that of their dietary lipids and the 3 fatty acid content was not systematically different for the two types of diets.Use of yeast plus capelin oil resulted in considerably higher numbers of both suspended and rotifer-associated bacteria than with use of algal diets. No differences were found for the algal species involved. The viability of the rotifers in a temperature and salinity test was far better in groups fed algae than in groups fed yeast plus capelin oil.The results indicated an overall better quality of rotifers cultivated with algae than for rotifers cultivated with yeast and oil. Questions related to proteins and the microflora of rotifers should be further emphasized in future research.     

Effect of algal diet and temperature on the biochemical composition of the rotifer, Brachionus plicatilis

Rotifers were fed on four species of unicellular algae (Dunaliella tertiolecta, Pavlova lutheri, Phaeodactylum tricornutum and Isochrysis galbana) at a range of temperatures. Measurements were made of growth rate, weight and level of total protein, carbohydrate and lipid. Large differences were found in rotifer weight and biochemical composition as a result of feeding and starvation. There were only minor differences attributable to the type of alga used as a food.     

Effect of unionized ammonia, viscosity and protozoan contamination on the enzyme activity of the rotifer Brachionus plicatilis

Substrates that are cleaved to yield fluorescent products can be used to quickly quantify enzyme activity in vivo with image analysis or in vitro with fluorometry. This study was carried out to determine whether enzyme activity in rotifers is useful for assessing the physiological condition of rotifers. Neonates of Brachionus plicatilis Müller hatched from cysts were exposed to a concentration series of unionized ammonia ranging from 0 to 9.8 p.p.m., increasing seawater viscosity relative to 1.17 to control sea water by the addition of methyl cellulose and the addition of the protozoan Euplotes sp. to a density of 40 mL^?1. Rotifer glucosidase and esterase activities decreased with increasing unionized ammonia and viscosity respectively. Activities of glucosidase and phospholipase decreased with increasing protozoan contamination. There was a significant relationship between enzyme activities and rotifer population growth. In vivo activities of certain rotifer enzymes can therefore serve as biomarkers for the rapid assessment of environmental stressors in rotifer mass cultures.