Lipid classes and fatty acid composition of rotifers (Brachionus plicatilis) fed two algal diets

Rotifers (Brachionus plicatilis), maintained on baker's yeast, were fed for 24h upon two algal diets, Isochrysis galbana (diet A) and Isochrysis galbana + Nannochloropsis gaditana (diet B). (These algal diets were selected for their potential use as essential fatty acid (EFA) boosters, taking into account the requirements of fish larvae). The effect of these algal diets on total lipid content, lipid classes and fatty acid composition was studied. The total lipid content increased after feeding upon both diets but no significant differences were found between the two types. Neutral lipid and polar lipid contents increased and a positive correlation was observed between the neutral lipids content of rotifers and that of the food supplied. However, the content of polar lipids in rotifers did not depend upon that of the diet. The increase in neutral lipid content was found to be higher in rotifers fed upon diet B, compared to diet A which increased the phospholipid content. Non-enriched rotifers contained only small amounts of polyenoic fatty acids, i.e. 18:3n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3, the contents of which increased significantly by feeding both diets. The EFA composition (20:4n-6, 20:5n-3 and 22:6n-3) of neutral lipids and phopholipids in rotifers reflected the EFA composition of each diet. Diet B-fed rotifers had the highest content in 20:4n-6 and 20:5n-3, whereas rotifers fed diet A and the highest 22:6n-3 content. The mixed diet I. galbana + N. gaditana enhanced substantially the composition of lipid classes i.e. neutral lipids and of n-3 PUFA of rotifers in comparison with Isochrysis or yeast diets.     

Effect of two microalgae concentrations on body size and egg size of the rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis>

The rotifer, Brachionus calyciflorus, was grown with two algae species (Chlorella sp. and Scenedesmus obliquus) at different concentrations (0.1, 1 and 10 × 10⁶ cells ml⁻¹). The body size (lorica biovolume) of individual rotifer and their egg size were measured when the populations were roughly in the exponential phase of population growth. The body size of the rotifers differed significantly (P < 0.05) among the two algae species used, however this effect was not observed for egg size. The body size of rotifers fed on higher densities of Chlorella sp. (10 × 10⁶ cells ml⁻¹) was significantly larger than for those fed on lower and medium densities (0.1 and 1 × 10⁶ cells ml⁻¹). Body size and egg size of rotifers fed with different amounts of Scenedesmus did not differ significantly. The egg size was significantly larger at higher food level of Chlorella. A significantly positive correlation was observed between the adult rotifer body size and their egg size.     

大型海藻龙须菜抽提液对褶皱臂尾轮虫生命表参数的影响

大型海藻富含多种活性物质,具有抗衰老等生物活性;轮虫是良好的潜在抗衰老研究模式生物。本研究以褶皱臂尾轮虫(Brachionus plicatilis)作为实验对象,研究了不同浓度的大型海藻龙须菜抽提液(0,250,500,750,1000 mg/L)和不同浓度的食物(蛋白核小球藻和普通小球藻)对褶皱臂尾轮虫生命表参数的影响。结果表明:与对照组相比,食物浓度为1.0×10~6个/mL蛋白核小球藻时,不同浓度龙须菜抽提液对轮虫产卵数、平均寿命、净生长率以及世代时间有显著促进效应(P0.05);轮虫平均产卵数及寿命在龙须菜抽提液浓度750 mg/L处达到最高,分别为16只和13.9d(P0.05)。食物浓度为2.0×10~6个/mL普通小球藻时,轮虫平均产卵数和寿命在抽提液浓度为500 mg/L处达到最高,分别为16只和13.6d(P0.05),轮虫平均寿命和净生长率均有显著提高(P0.05)。相同龙须菜抽提液浓度下,食物浓度为1.0×10~6个/mL蛋白核小球藻下轮虫的净生长率、世代时间均显著高于食物浓度为2.0×10~6个/mL蛋白核小球藻培养的轮虫(P0.05);食物浓度为2.0×10~6个/mL时,普通小球藻培养轮虫的净生长率和世代时间均显著高于蛋白核小球藻实验组(P0.05)。交互作用分析显示,龙须菜抽提液与小球藻的交互作用对褶皱臂尾轮虫的内禀增长率有显著影响(P0.05)。研究结果表明,大型海藻龙须菜抽提液对褶皱臂尾轮虫的生长与生殖有促进作用,延长轮虫寿命。     

Biotechnology and aquaculture of rotifers

Biotechnology can be defined as any technology that involves living organisms or their derivatives. In applying this definition to rotifers, we focus on their contribution in culturing of early larval stages of marine fish. After almost four decades of marine fish culture in captivity, the success of this worldwide industry is still quite dependent on mass culture of the species Brachionus plicatilis and B. rotundiformis. In mass culture, the rotifers are continuously driven to reproduce at high rates, in relatively extreme environmental conditions of high population density and high loads of organic matter. Therefore, the success of mass cultures and future improvements in these systems relies on a close interaction between basic and applied studies of rotifers. In the present review, we will attempt to analyze why rotifers are suitable for early life stages of fish and to describe, in general, methodologies that have been devised for reliable supply of rotifers in large quantities. Problems associated with rotifer production, nutritional quality and effect on fish health and nutrition, will be discussed. Research on B. plicatilis and B. rotundiformis has increased enormously during the past three decades and these two species are the best-studied rotifers so far. While much of the research on these species is directed or devoted to the needs of aquaculture industry, they are also used as models for addressing basic biological questions, due to the relative ease of culture and their availability. Studies on feeding, pheromones, speciation in rotifers, the occurrence and putative hormones involved in sexual and asexual reproduction and production of resting eggs, are few examples of such studies. Rotifers will probably maintain their role as food organism for fish larvae, in spite of attempts to replace them with more accessible formulated food. Development of new culture methods that will improve the nutritional quality and production efficiency of rotifers may result in more diversified and flexible tasks for these organisms in aquaculture.     

Presence and development of populations of the introduced brown alga Sargassum muticum in the southwest Netherlands

Of the three Brachionus species used in aquaculture, Brachionus rubens, B. calycilorus and B. plicatilis, the latter is most widely used in raising marine fish and shrimp larvae due to its tolerance to the marine environment. In freshwater aquaculture the use of B. rubens and B. calycilorus is limited, probably because inert food products are readily available as feed for freshwater larvae.The rotifer Brachionus plicatilis is used in large numbers as the first food organism in intensive cultures of marine fish and shrimp larvae. An adequate supply of these rotifers relies on mass cultures. The reproductive rate of rotifers in these cultures depends on food quality and quantity, salinity, temperature and pH of the medium. Removal of waste products from culture tanks leads to higher and more efficient production of rotifers over extended periods of time. Rotifers have to be enriched with polyunsaturated fatty acids, which are essential for proper development and survival of marine fish and shrimp larvae.The future use of preserved rotifers and their resting eggs may help to overcome unforeseen failures of live cultures and may lead to more efficient use of these organisms in raising freshwater and marine fish and shrimp larvae.     

Production and nutritional quality of the rotifer Brachionus plicatilis fed marine Chlorella sp. at different cell densities

The effect of different cell densities of marine Chlorella sp. on the growth rate, doubling time and production of the rotifer Brachionus plicatilis was investigated. A significant increase in rotifer production was achieved at a density of 50 × 10⁶ Chlorella cells ml^–1. The nutritional quality of rotifers grown at different concentrations of Chlorella is discussed.     

A method for the detection of Issatchenkia orientalis in a baker's yeast factory

Summary A method for the detection of Issatchenkia orientalis (Candida krusei), a contaminant of bakers' yeast, is described. Nine different liquid medium types were compared and maximum specific growth rates for the yeast contaminant were determined in each medium. Issatchenkia orientalis grew fastest in malt extract broth (0,37 h^-1) and potato dextrose broth (0,36 h^-1). Five antibiotics were tested for selective inhibition of seven different bakers' yeast strains in malt extract broth. Nystatin was the only antibiotic tested that inhibited the growth of all the bakers' yeast strains used, but did not affect the growth of I. orientalis.     

Intensive rotifer cultures using chemostats

Continuous production of the rotifer Brachionus plicatilis rotundiformis (S-type) in an intensive chemostat culture system has been investigated. The production dynamics of rotifers in relation to different flow rates and feed regimes show that the growth rate and production depends on the type of algal feed and flow rate utilized in the culture system. It was possible to achieve a mean production of up to 318.84 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 6 1 h^–1 in 100 1 chemostats and up to 261.21 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 40 1 h –1 while using 1 m³ capacity rotifer chemostats as production units. The 3 fatty acid composition of rotifers while using Chlorella and Nannochloropsis in the culture system has been described. The results of this investigation show that the rotifer productivity in the continuous culture system is considerably higher than in any of the conventional culture systems described to date for aquacultural purposes.This research was financed by the Kuwait Foundation for the Advancement of Sciences (KFAS), Kuwait, under a contract research project code 86-04-02.     

Characterization of the bacterial flora of mass cultivated Brachionus plicatilis

Bacterial density and composition in association of mass cultivated rotifers (Brachionus plicatilis, SINTEF-strain) was investigated, during experimental conditions identical to the procedures used for preparing rotifers as live food for marine cold water fish larvae. These procedures include cultivation, enrichment with squid meal and acclimation to low temperature by storage of the rotifer culture at 6 °C. Large variations were observed in the number of rotifer associated (1.8–7.6 · 10³ colony forming units per rotifer^–1) and free-living (0.6–25 10⁷ cells·ml^–1) bacteria. An increase of 50–150% in the bacterial number was normally observed after feeding the rotifer with squid meal, but after three days of acclimation at 6 °C, the bacterial numbers decreased to the initial level.After enrichment of the cultures with squid meal, the similarity in the composition of the bacterial flora between the rotifers and water was reduced. However, acclimation of the culture at 6 °C resulted in better agreement of the rotifer associated flora and that in water. Enrichment of the cultures induced a shift in the bacterial composition from Cytophaga/Flavobacterium dominance to Pseudomonas/Alcaligenes dominance. The bacterial flora of the rotifer cultures are dominated by presumably opportunistic species after enrichment, which may have detrimental effects when rotifers are fed as live food to marine fish larvae.     

Clearance rates of sessile rotifers: in vitro determinations

We measured laboratory clearance rates of 10 rotifer and one unidentified bryozoan species from 3 different lakes using ³²P labeled algae (Chlamydomonas) or yeast (Rhodotorula). Clearance rates for all rotifers fed yeast ranged from < 2.0 to > 260 µl · animal^–1 · h^–1 depending on species. The in vitro clearance rates of two sessile rotifers (Ptygura crystallina and P. pilula) were not significantly different from previously measured in situ rates (Wallace and Starkweather 1983). Clearance rates for 5 rotifers fed algae ranged from < 5.0 to > 90.0 µl · animal^–1 · h^–1. Ptygura beauchampi, P. crystallina, P. pilula, Floscularia conifera, and F. melicerta ingested both cell types but their clearance rates varied substantially among species and between cell types. There was a substantial time-dependent loss of 32P from formalin-fixed animals (Sinantherina socialis) awaiting processing. This loss stabilized at approximately 20 hours and was estimated to be about 40% of the initial ingested label. Clearance rates for the bryozoan fed yeast or algae were highly variable, ranging from < 1.0 to > 3 000 µl · animal^–1 · h^–1.     

Enriching Rotifers with “Premium” Microalgae. Nannochloropsis gaditana

The nutritive quality of Nannochloropsis gaditana cultured semicontinuously with different daily renewal rates was tested as a diet for short-term enrichment of the rotifer Brachionus plicatilis. After 24 h, dramatic differences in the survival, dry weight, and biochemical composition of the rotifers depending on the renewal rate of microalgal cultures were observed. Survival after the feeding period increased with increasing renewal rates. Rotifers fed microalgae from low renewal rate, nutrient-deficient cultures showed low dry weight and organic contents very similar to those of the initial rotifers that were starved for 12 h before the start of the feeding period. On the contrary, rotifers fed nutrient-sufficient microalgal cells underwent up to twofold increases of dry weight and protein, lipid, and carbohydrate contents with regard to rotifers fed nutrient-depleted N. gaditana. Consequently, feed conversion rate decreased in these conditions, indicating a better assimilation of the microalgal biomass obtained at high renewal rates. No single microalgal biochemical parameter among those studied can explain the response of the filter feeder. Similarly to gross composition, EPA and n-3 contents in rotifers fed microalgae from nutrient-sufficient cultures were double than the contents found in rotifers fed nutrient-limited microalgae. In addition, very high positive correlations between the contents of EPA and n-3 in N. gaditana and B. plicatilis were observed. These results demonstrate that selecting the appropriate conditions of semicontinuous culture can strongly enhance the nutritional value of microalgae that is reflected in the growth and biochemical composition of the filter-feeder even in short exposure periods.     

Use of freeze-dried microalgae for rearing gilthead seabream, Sparus aurata L., larvae. II. Biochemical composition

The objectives of this study were to test whether freeze-dried microalgae are nutritionally adequate for rearing rotifers as food for gilthead seabream larvae. The elemental composition (C, N, H) and fatty acid composition were analysed in larvae of gilthead seabream, Sparus aurata L., rotifers Brachionus plicatilis and Brachionus rotundiformis and freeze-dried microalgae Nannochloropsis oculata. Four larval feeding treatments were tested: (A) larvae fed rotifers cultivated with freeze-dried microalgae and daily addition of freeze-dried microalgae to the larval tanks; (B) larvae fed rotifers cultivated with freeze-dried microalgae and daily addition of live microalgae to the larval tanks; (C) larvae fed rotifers cultivated with freeze-dried microalgae, without addition of microalgae to the larval tank and (D) larvae fed rotifers cultivated with live microalgae and daily addition of live microalgae to the larval tanks. No significant differences were observed between the biochemical composition of larvae with treatment A (with freeze-dried microalgae) and the composition of larvae in treatment D that were obtained with the acceptable methods for culture systems (with live microalgae).     

Overexpression of bacterioferritin comigratory protein (Bcp) enhance viability and reduced glutathione level in the fission yeast under stress

The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCPlO. The bcp ⁺ mRNA level in the pBCPlO-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCPIO exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.     

Isolation of ethanol-tolerant mutants of yeast by continuous selection

Summary Mutants of Saccharomyces uvarum, 5D-cyc with increased tolerance to ethanol have been isolated by a continuous selection technique which allows the culture itself to determine the intensity of selection via a feedback control circuit. The output of CO₂ from a continuous culture of the yeast was monitored using an infrared analyser and the signal from that analyser fed to a potentiometric controller which regulated the introduction of a concentrated ethanol solution into the culture vessel. The frequency of ethanol addition to the culture thus increased as the tolerance of the organisms improved.The use of this system permitted the selection of mutants of yeast which were viable in the presence of 12% w/v ethanol and which showed higher fermentation rates (as measured by CO₂ production) than the wild-type in the presence of 10% w/v ethanol and above. The technique of continuous selection with feedback should be generally applicable to the isolation of mutants of any microorganism to improved tolerance to any inhibitory condition of either its physical or chemical environment.     

Biological evaluation of frozen zooplankton as food for milkfish (Cbanoschanos) fry

Milkfish fry with an average standard length and weight of 13.88 mm and 3.95 mg, respectively, were reared for 30 days using live and frozen Moina macrocopa and Brachionusplicatilis at feeding densities of 10–20 individuals per ml. Growth, survival and yield were used as indicators of the overall performances of the various treatment groups. Fry fed live M. macrocopa showed gains (both length and weight), growth and survival rates and yields significantly higher than fry fed with other treatment groups (P < 0.05). However, significant reductions in growth and survival rates resulted when fry were fed frozen M. macrocopa. On the other hand, there were no significant differences in growth and survival rates (P < 0.05) in fry fed live or frozen B. plicatilis. The results of the current study showed that although milkfish fry could be grown successfully using B. plicatilis, feeding with live Moina significantly improved growth, survival rate and yield (P lt; 0.05). Frozen Moina was found to be unsuitable as a feed for rearing milkfish fry because it reduced growth rates and increased mortality. Comparisons between live and frozen rotifers have proven the suitability of frozen rotifers as feed for rearing milkfish fry. By freezing surplus rotifers this would permit short term storage in anticipation of high hatchery demand and overcome any unpredictable failures with live cultures.     

Laboratory culture of fairy shirmps using baker's yeast as basic food in a flow-through system

We designed and standardized a culture method for freshwater anostracans using diets free of live algae.Thamnocephalus platyurus andBranchinecta lindahli were used as test organisms.We used baker's yeast as basic food and added inert particles (clay or amorphic silicium dioxide) to improve the digestion of the yeast. A flow-through culture system was used, according to a fixed feeding schedule, to supply separately, culture medium (tap water), food, and inert particle suspensions. Three variants with baker's yeast as basic, food were compared for survival, growth, and reproduction. A diet of solely baker's yeast (diet 1) or baker's yeast supplemented with vegetal oil containing ß-carotene (diet 2) was unsuitable for reproduction ofT. platyurus. Cyst production was only achieved when diet 2 was supplemented with fish oil andSpirulina powder (diet 3). This suggests that not only a digestibility problem, but also nutritional deficiencies are present in baker's yeast.     

Nutritional effect of freshwater Chlorella on growth of the rotifer Brachionus plicatilis

Mass production of Brachionus plicatilis is usually accomplished by feeding so-called marine Chlorella (Nannochloropsis oculata) to the rotifers in marine fish hatcheries. If the marine Chlorella are in short supply, baker's yeast is usually used as a supplementary food. Recently, a condensed suspension of freshwater Chlorella (Chlorella vulgaris, k-22) was commercially developed as another supplementary food. We have evaluated the dietary value of this freshwater Chlorella for growth of the rotifer by means of individual and batch cultures. Rotifers cultured with the freshwater Chlorella suspension under almost bacteria-free conditions, showed very suppressed growth. However if the Chlorella was supplemented with vitamin B₁₂ by adding the vitamin solution into the suspension or by culturing the Chlorella in a medium containing vitamin B₁₂, the nutritional value of freshwater Chlorella was greatly improved and almost at the same level as that of marine Chlorella. Condensed Chlorella may therefore be effective as a supplementary food if vitamin B₁₂ is supplied.     

Inulinase production by a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the crude inulinase

Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60°C. The optimal medium for inulinase production was seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28°C and 170 rpm. Under the optimal conditions, over 60 U ml⁻¹ of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.     

Expression of the oligopeptide transporter, PepT1, in larval Atlantic cod (Gadus morhua)

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract.     

Physiological adaptations in the survival of rotifers (Brachionus plicatilis,O. F. Müller) at low temperatures

The biology of the rotifer Brachionus plicatilis has been studied extensively in recent years, due to its importance in aquaculture. Today, the culture of several marine fish species relies completely on the daily production of live rotifers.In the present paper we explore the conditions that facilitate maintaining live rotifers for extensive periods at low temperatures. In addition to its possible contribution in providing reserve stocks for commercial application, these studies may be of ecological importance. They could explain some of the physiological adaptive mechanisms that are involved in the survival of rotifers under adverse environmental conditions.Experimental results showed relatively high survival rates (82–85%) in rotifers that were cultured at 25 °C and exposed later to -1 °C for 12–14 days. During this period, rotifers were kept without food and their media were not changed. The survival was found to depend on the rotifer culture conditions, prior to exposure to -1 °C. These included the type of food fed to rotifers (yeast or algae), the salinity in which they were cultured, and an essential acclimation period of 2–6 day at 4 °C or 10 °C. The acclimation period was associated with the synthesis of at least one specific protein and accumulation of lipids. Profiles of protein synthesis in rotifers incubated at 10 °C revealed a 94 kD protein, which did not appear in rotifers cultured at 25 or 37 °C. Immunoisolation, using a polyclonal antibody that was prepared against HSP60, revealed that this protein was synthesized in rotifers kept at 10, 25 or 37 °C. However, this antibody did not react with the 94 kD peptide.In addition, rotifers kept at 10 °C accumulated substantial amounts of lipids, including eicosapentaenoic acid (EPA), which is found in the algae fed to them. These results support the hypothesis of specific adaptations to survival at low temperatures during an acclimation period.