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.     

Rotifers as food in aquaculture

The rotifer Brachionus plicatilis (O.F. Muller) can be mass cultivated in large quantities and is an important live feed in aquaculture. This rotifer is commonly offered to larvae during the first 7–30 days of exogenous feeding. Variation in prey density affects larval fish feeding rates, rations, activity, evacuation time, growth rates and growth efficiencies. B. plicatilis can be supplied at the food concentrations required for meeting larval metabolic demands and yielding high survival rates. Live food may enhance the digestive processes of larval predators. A large range of genetically distinct B. plicatilis strains with a wide range of body size permit larval rearing of many fish species. Larvae are first fed on a small strain of rotifers, and as larvae increase in size, a larger strain of rotifers is introduced. Rotifers are regarded as living food capsules for transferring nutrients to fish larvae. These nutrients include highly unsaturated fatty acids (mainly 20: 5 n–3 and 22: 6 n–3) essential for survival of marine fish larvae. In addition, rotifers treated with antibiotics may promote higher survival rates. The possibility of preserving live rotifers at low temperatures or through their resting eggs has been investigated.     

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.     

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).     

Mariculture in Israel – past achievements and future directions in raising rotifers as food for marine fish larvae

Marine fish production is now being carried out afteralmost two decades of research. The production ofseabream (Sparus aurata), which reached over 750tons in 1995, is expected to reach an annualproduction ranging between 4000 - 12 700 metric tonsby year 2010. The anticipated introduction of newspecies and its expansion to the Mediterranean shoreline will help in leading the increased maricultureproduction. Two marine fish hatcheries that operatetoday in Israel produce 7 million fingerlings a year.Traditionally, aquaculture in Israel raises fish ininland freshwater ponds and irrigation reservoirs. Inaddition, Lake Kinneret, the only freshwater lake inIsrael, is stocked yearly with juvenile fish raised inlocal hatcheries (tilapia) or imported fromMediterranean countries (mugil). While culture offreshwater teleost species (carp) was introduced morethan fifty years ago, mariculture started on acommercial scale less than 5 years ago. The limitedsupply of freshwater will accelerate the futureculture of marine species.The bottleneck of almost all marine finfish productionlies in obtaining adequate numbers of fingerlings, dueto their high mortality at early life stages. Theproduction is hindered by inadequate supply of food toearly larval stages which require live food.Development of technologies in Israel for masscultivation of food chain organisms including algae,rotifers and brine shrimp followed their developmentin other parts of the world, most notably thoseachieved in Japan. The local commercial scaleproduction of rotifers relies on several batch orsemi-continuous cultures in conical or flatbottomrectangular containers that supply daily 0.6-4billion rotifers in each hatchery. Originally arelatively large local Brachionus plicatilisstrain was used, but later smaller B.rotundiformis strains were introduced, resulting ina mixture of undefined strains. The incorporation ofalgae (Nannochloropsis sp.) generated in highyield raceways contributes to the reliability ofrotifer cultures. Algae are supplied directly from theraceways or centrifuged and stored as a frozen pasteuntil required in the hatchery. The current dependablesupply of live cultures reduces the need for preservedstocks of rotifers, either as resting eggs or keptalive at low temperatures. To the fish grower,rotifers are live food capsules that deliver essentialnutrients (e.g. long chain unsaturated fatty acids)for growth and survival of fish larvae. Research aimedat replacing live food with chemically definedmicrodiets could reveal physiological principles inprey recognition and digestion of food by marine fishlarvae.     

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.     

The use of marine yeast (Candida sp.) and bakers' yeast (Saccharomyces cerevisiae) in combination with Chlorella sp. for mass culture of the rotifer Brachionus plicatilis

Use of marine yeast and bakers' yeast in combination with Chlorella sp. for the large-scale production of the rotifer Brachionus plicatilis was investigated. The culture density of marine yeast fed rotifers was significantly higher than rotifers fed bakers' yeast. Rotifer production was significantly higher and the doubling time was lower for marine yeast fed rotifers than for bakers' yeast fed rotifers. It appears that the addition of marine yeast to the feed enhances the birth rate and overall production of rotifers in the culture system. The nutritional quality of rotifers is discussed.     

Feeding biology of two brachionid rotifers: Brachionus quadridentatus and Brachionus plicatilis

Brachionus quadridentatus and Brachionus plicatilis were examined in laboratory cultures to investigate their (1) food requirements and (2) ability to select particles on the basis of size. Growth experiments showed that the Chlorophyceans Nannochloris sp. and Chlorella vulgaris possess good nutritional value for these rotifers. However, B. quadridentatus declined in cultures with Microcystis firma, detritus of Enteromorpha sp. or Pseudomonas sp. (bacteria) as a sole source of food, thus indicating that these foods were inadequate. Utilization of latex microspheres by these two species indicate that they respond to particle size in different ways: B. quadridentatus had a higher selectivity index for 3 and 5 µm beads, while B. plicatilis had a higher selectivity index for 2 µm beads.     

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.     

Studies on Brachionus (Rotifera): an example of interaction between fundamental and applied research

The genus Brachionus has been the main subject of studies reported in about 1000 papers published since 1950. About three-fourths of these deal with Brachionus plicatilis and B. rotundiformis and are mainly related to their use as prey for aquatic organisms. Also abundant, but to a lesser extent, are studies on B. calyciflorus, many of which are concerned with aquatic ecotoxicology. These studies constitute an interesting interaction between fundamental and applied research. For example, advances in fundamental biology have been applied to improve the production of rotifer biomass. Alternatively, new perspectives in fundamental research on rotifers have emerged while solving technical and biological problems related to the rearing of aquatic animal larvae. This review describes some aspects that have shown a significant advance due to such interaction between fundamental and applied research on rotifers, e.g. growth conditions, biochemical composition and morphotypes.     

Effect of Food Quantity and Quality on Population Growth Rate and Digestive Activity in the Euryhaline Rotifer Brachionus plicatilis Müller

We investigated the nutritional effects of both food quantity and quality on Brachionus plicatilis. Decomposition of particulate and dissolved organic matter by rotifer digestive enzymes play a crucial role in rotifer nutrition. Among other enzymes, rotifers produce phosphatases, non‐specific enzymes that allow for the release of orthophosphate from a variety of organic phosphorus compounds. Phosphatase saturation was measured in B. plicatilis homogenates using the spectrofluorimetric method. We examined population growth rate, reproduction and phosphatase activity in the homogenate of rotifers (PA_RH) fed by nutrient‐replete algal food supplied at different quantities. Population growth rate, number of eggs per individual and PA_RH were affected by food quantity. Growth rate and number of eggs per individual significantly increased in rotifers fed by food supplied at the highest quantity. The highest population growth rate was reached by rotifers fed by nutrient‐replete food, while it did not significantly differ between rotifers fed on nitrogen (N)‐depleted and phosphorus (P)‐depleted food. The number of eggs per individual was more affected by N than P supply. PA_RH and rotifer RNA content were not influenced by different food quality. The results indicate that B. plicatilis is not able to regulate its digestive apparatus in terms of efficiently getting access to essential nutrients when scarce, but do this when nutrient‐replete food is available in different quantity. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production. Received April 12, 1999; accepted July 29, 1999.     

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.     

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.     

Chemoreception and vertical movement in planktonic yolk-sac larvae of red sea bream Pagrus major

The capability of planktonic yolk-sac larvae of red sea bream Pagrus major in detecting food was examined in the laboratory to ascertain basic knowledge on the early life history of this marine fish. After infrequent vertical burst swimming followed by slight rising or sinking, the larvae remained motionless within thin layers of concentrated food extract (rotifer, Brachionus plicatilis). At the moment of hatching, the larvae already have receptor cells with several cilia arranged radially in their open nostrils. Thus it is likely that by means of their vertical movement they are capable of sensing the thin food patch layer. We suggest that planktonic larvae of Pagrus major are capable of detecting and remaining within food patches even before the onset of feeding. The onset of food detection in the earlier stages may be, to some extent, the more efficient strategy for larval survival and growth because this ability could contribute to a reduction in energy consumption.     

Toxicity and haemolytic activity of a newly described dinoflagellate,Heterocapsa bohainensis to the rotifer Brachionus plicatilis

The algae Heterocapsa bohaiensis is a newly described species of dinoflagellate associated with Penaeus japonicus and larvae of Eriocheir sinensis in a coastal pond of Liaodong Bay China. The rotifer Brachionus plicatilis is used as live feed for aquaculture organisms including prawns and crabs larvae. To evaluate the potential toxicity of H. bohaiensis, the effects on Brachionus plicatilis and haemolytic activity were investigated in this study. The results showed that H. bohaiensis had significant toxic effect on B. plicatilis, and this effect was dependent on the cell concentration. Significant rotifer growth suppression was observed in the ruptured cells of H. bohaiensis with ultrasonic. Relatively similar rotifer mortalities were induced both in the light and in the dark. Interestingly, haemolysis to erythrocytes was also caused in a cell density-dependent and time-dependent manner, which meant the results of haemolytic activity were consistent with the toxicity. Therefore, haemolytic toxins were considered to be involved in the toxic mechanism of H. bohaiensis against rotifers. Then, the concentrations of calcium were measured in the mastax, stomach and ovary of B. plicatilis. Obviously increased fluorescence intensity was found in the stomach, which indicated the alteration of calcium homeostasis and membrane permeability after ingesting H. bohaiensis. These results implicated haemolytic activity as a causative factor linked to the toxicity of H. bohaiensis against B. plicatilis. The results contributed to research the production and control of H. bohaiensis toxins.     

Life history characteristics of Brachionus plicatilis (rotifera) fed different algae

A detailed study of the life history of the rotifer Brachionus plicatilis was done at 20 °C, 20 ppt salinity, and 90 mg C 1^–1 food concentration. Rotifers were grown individually in culture plate wells (150 µl culture volume) and fed Isochrysis galbana Tahiti, Tetraselmis sp., Nannochloris atomus, or a l : 1 mixture (weight) of two of the algae. Observations were made every 2–8 hr and rotifers were sized and transferred to new food daily. A total of 19 different parameters were compared. Rotifers fed Isochrysis averaged 21 offspring per female, a 6.7 day reproductive period, a lifespan of 10.5 days and a mean length of 234 µm. After Isochrysis, the foods giving the highest growth, survival, and reproduction in decreasing order were Isochrysis + Nannochloris, Nannochloris, Isochrysis + Tetraselmis, Tetraselmis + Nannochloris, and Tetraselmis. Although the small volume culture system used in this study seems appropriate for studying life history of B. plicatilis, the results cannot always be directly applied to larger cultures.     

Induction of sexual reproduction and resting egg production in Brachionus Plicatilis reared in sea water

Brachionus plicatilis raised in our laboratory in sea water reproduces asexually even under high crowding conditions (at least 40 individuals per ml). Amictic females were induced to produce mictic females, males and resting eggs by reducing the concentration of the sea water culture medium. Mictic females and males appeared predominantly among the progeny produced by the amictic females during 4 days following their transfer into 25% sea water. Resting eggs appeared first 5–12 days after the onset of the experiment. Following the disappearance of males, the culture consisted of amictic females.Resting eggs produced by the method described above may be preserved for at least three months at –14°C or by desiccation at room temperature. Under the appropriate experimental conditions, resting eggs hatch into amictic females. Since B. plicatilis is one of the most commonly used food sources of fish larvae in aquaculture, the methods reported here may offer an easy and versatile way of preserving rotifer culture stock to be used on demand.     

Particle size dependent feeding by the rotifer Brachionus plicatilis

Size selective feeding by Brachionus plicatilis was investigated with algae and bacteria (0.3–3.5 µm) and mono-disperse latex beads (0.3–3.0 µm) in short term feeding experiments. B. plicatilis demonstrated maximum clearance rate of particles with diameter 2µm, but particles with diameter down to 0.3 µm were also ingested. The clearance rate of bacteria was 15–55% of that obtained for optimal sized particles (2 µm), and was related to particle size. The relative reduction in retention of particles with diameter < 2 µm was more pronounced for latex beads than for natural food particles, suggesting other mechanisms than size to be important for the particle retention by the rotifer. This is emphasized by the fact that the clearance rates were much lower for latex beads than for natural food particles of comparable size. Efficient retention of bacteria was observed for rotifers in poor physiological condition, i.e. rotifers with low maximum clearance rate. This may reflect a strategy to optimize energy utilization by reducing locomotion costs and increasing energy intake.The results indicate that B. plicatilis has a low to medium ability to feed on bacteria. In natural ecosystems, its importance as a bacterial grazer is of limited importance. At high population densities, such as in live feed cultures, the rotifer may, however, efficiently remove bacteria from the culture.     

Feeding kinetics of Brachionus plicatilis fed Isochrysis galbana

Clearance and ingestion rates of Brachionus plicatilis were measured using ¹⁴C-labeled Isochrysis galbana Tahiti. Experiments were conducted at 20–22 °C, 20 ppt salinity, and algal concentrations ranging from 0.13–64 mg C 1^–1. Clearance rates were constant and maximal at concentrations <2 mg C 1^–1, with maximum rates ranging from 3.4–6.9 µl ind.^–1 hr^–1. The ingestion rate varied with food concentration, and was described by a rectilinear model. The maximum ingestion rate varied considerably, and was dependent on the growth rate of the rotifers. Depending on the pre-conditions, B. plicatilis ingested about 0.5 to 2 times its body carbon per day at saturating food concentrations.