Insulin-like Growth Factor Signaling Pathway Involved in Regulating Longevity of Rotifers

Rotifers have been used to study the mechanisms of ageing for more than a century, but the underlying molecular basis of ageing in rotifers is largely unknown. The insulin/insulin-like growth factor (IGF-1) signaling pathway has been found to regulate the lifespan of evolutionarily distinct eukaryotes from yeast to mammals. We therefore assume that the insulin/IGF-1 pathway is a candidate for regulating the rotifer’s lifespan. Accordingly, we examined the action of an inhibitor to PI3-kinase involved in the pathway for the rotifer Brachionus plicatilis O. F. Müller. This kinase was first discovered as age-1 to regulate the longevity of Caenorhabditis elegans. As expected, the inhibitor treatment resulted in the extension of lifespan by 30% compared to the reference group without the treatment, whereas reproductive characters were not apparently changed. These results were consistent with those observed in C. elegans, suggesting that the lifespan of B. plicatilis is likely to be regulated by the signaling pathway involving PI3-kinase.     

Molecular and expression analysis of manganese superoxide dismutase (Mn–SOD) gene under temperature and starvation stress in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species. We cloned cDNA encoding SOD activated with manganese (Mn–SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of Mn–SOD was 1,016 bp and had a 669 bp open reading frame encoding 222 amino acids. The deduced amino acid sequence of B. calyciflorus Mn–SOD showed 89.1, 71.3, and 62.1 % similarity with the Mn–SOD of the marine rotifer Brachionus plicatilis, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, respectively. The phylogenetic tree constructed based on the amino acid sequences of Mn–SODs from B. calyciflorus and other organisms revealed that this rotifer is closely related to nematodes. Analysis of the mRNA expression of Mn–SOD under different conditions revealed that expression was enhanced 5.6-fold (p < 0.001) at 30 °C after 2 h, however, low temperature (15 °C) promoted Mn SOD temporarily (2.5-fold, p < 0.001) and then decreased to normal level (p > 0.05). Moderate starvation promoted Mn–SOD mRNA expression (p ₁₂ < 0.01, p ₃₆ < 0.05), which reached a maximum value (15.3 times higher than control, p ₂₄ < 0.01) at 24 h. SOD and CAT activities also elevated at the 12 h–starved group. These results indicate that induction of Mn–SOD expression by stressors likely plays an important role in aging of B. calyciflorus.     

Molecular genetics of rotifers: preliminary restriction napping of the mitochondrial genome of Brachionus plicatilis

Methods are presented to extract and purify mitochondrial DNA from the rotifer Brachionus plicatilis. The mtDNA obtained is of sufficient purity for digestion with restriction endonucleases. EcoR I restriction patterns are presented for 4 geographically separated clones. A restriction map based on digestion with 5 different restriction enzymes is included for one of these clones. Finally, use of mtDNA analysis for studies on the population structure and biogeography of rotifers is discussed.     

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)     

Interactions between red tide microalgae and herbivorous zooplankton: effects of two bloom-forming species on the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> (O.F. Muller)

Experiments were carried out to investigate interspecific interactions between the rotifer Brachionus plicatilis and two harmful algal bloom (HAB) species using single and mixed culture methods. B. plicatilis populations and the growth of two algae were compared at different algal cell densities. The results demonstrate that B. plicatilis obtained sufficient nutrition from Alexandrium tamarense to support net population increase. When exposed to a density of 8 × 10⁴ cells ml⁻¹ A. tamarense, the number of B. plicatilis increased faster than it did when exposed to other four algal densities (16 × 10⁴, 24 × 10⁴, 32 × 10⁴, and 40 × 10⁴ cells ml⁻¹). Cell densities of A. tamarense decreased due to the grazing of B. plicatilis. In contrast, Heterosigma akashiwo had an adverse effect on the B. plicatilis population and its growth was largely unaffected by rotifer grazing. In this case, the B. plicatilis population decreased and H. akashiwo grew at a rate similar to that of a control without addition of rotifers. Mixed culture experiments showed that A. tamarense could partly counteract the effect of H. akashiwo in limiting the rate of population increase of rotifer. In addition, the effect of different initial cell densities on interspecific competition between A. tamarense and H. akashiwo in mixed culture(s) was also investigated. The results show that A. tamarense competed very successfully when the inoculation proportions of A. tamarense and H. akashiwo were 40:5 and 40:30. Handling editor: D. Hamilton     

A novel growth-promoting protein in the conditioned media from the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> at an early exponential growth phase

We confirmed the existence of growth-promoting substances in the conditioned media (CM) from the rotifer Brachionus plicatilis at an early exponential growth phase and isolated a novel protein with a growth-promoting activity from the crude extract (CE) of rotifer cells. CM was prepared from the culture media where rotifers had been cultured at an early exponential growth phase and filtered through a 0.22-μm filter membrane. The growth-promoting activity was determined using rotifers in CM for 5 days. As a result, the increase of rotifers added with CM was significantly higher than that of the control in artificial seawater (P < 0.001). Moreover, the growth-promoting activity of CM was dose-dependent and inactivated by heat treatment at 80°C for 60 min. Meanwhile, CM filtered through a <10 kDa ultrafiltration membrane showed a low activity, whereas proteinase K treatment resulted in a complete inactivation. These results suggest that the rotifer secrets growth-promoting proteins into CM. CE also contained a protein with the activity and properties similar to those found in CM. Then, CE was subjected to purification of a growth-promoting protein for convenience using various types of chromatography after fractionation with 30–80% saturated ammonium sulfate. Subsequently, a protein with an approximate molecular weight of 25000 was isolated, and its N-terminal amino acid sequence was determined to be PAVVDFTAVWFGPLQMIKP. An orthologue was found in the EST database of B. plicatilis, the full sequence of which showed about 50% identity to the corresponding regions of thioredoxins from other organisms.     

Screening Methods for Improving Rotifer Culture Quality

We studied whether the selection of rotifer B. plicatilis strain (Japanese, Russian or Australian), as well as the addition of gamma-aminobutyric acid (GABA) to the culture water, are useful in stabilising rotifer cultures. We examined the effect of a combination of the following stressors: unionized ammonia (2.4 mg l⁻¹), contamination by protozoa Euplotes sp. (10 cells ml⁻¹) and addition of methyl cellulose to increase the culture water viscosity at 15 cp. Rotifer reproductive tests and enzyme activity measurements (glucosidase) were conducted to determine the effect of the treatments. All tests were conducted at 25 °C and rotifers were fed Nannochloropsis oculata at 7 × 10⁶ cells ml⁻¹. The combined effects of the stressors caused a significant decrease in lifespan, fecundity and glucosidase activity. The effect of the stressors on reproductive characteristics and glucosidase activity could be neutralized if rotifers were treated with GABA.     

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.     

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.     

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.     

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.     

Axenic culture of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> using antibiotics

The rotifer Brachionus plicatilis culture is composed of complex microcosms including bacteria, protozoans, algae, and fungi. Previous studies reported methods to establish axenic rotifer cultures, but further refinement of these techniques is needed, for molecular biological research which requires pure culture to isolate nucleic acids from rotifers only. In order to render rotifer culture axenic, we tested five antibiotics: ampicillin (Amp), chloramphenicol (Cp), kanamycin (Km), nalidixic acid (Na), and streptomycin (Sm) at 30–100 μg/ml. Except for Cp, which reduces rotifer reproduction, all other antibiotics at the tested concentrations did not affect rotifer reproduction or show any toxic effects. A rotifer disinfection method was finally established by treating the resting eggs with 0.25% (w/v) sodium hypochlorite (NaOCl) for 3 min, washing with sterilized sea water, and then exposing the neonates to an Amp, Km, Na, and Sm mixture. Using four nutrient media, we confirmed that this protocol renders the rotifer culture bacterial and fungus free. The axenic rotifer culture generated here is useful not only for genetic analysis of Brachionus plicatilis, but for studying the rotifer life cycle without bacterial influence.     

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.     

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.     

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.     

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.     

Rotifer nutrition using supplemented monoxenic cultures

The evolution of rotifer feeding/ nutritional studies is discussed together with their relevance to ecological observations. Aseptic conditions and initially synxenic cultures are regarded as a basis for nutritional work. The marine rotifer Encentrum linnhei requires the amino acid dl-tryptophan as a supplement to the food-alga Brachiomonas submarina. Observations on feeding rotifers in natural water samples, together with the morphology of their feeding mechanisms, show Encentrum to be an omnivore; a natural source of tryptophan is suggested. Vitamin B₁₂ and thiamine requirements of Encentrum and Brachionus plicatilis are examined and evidence shown for the quantitative control of the former vitamin by the rotifer's food algae. Axenic cultivation of rotifers is discussed and restricted growth of Brachionus reported under such conditions.     

Studies on life history characteristics of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> O. F. Müller (Rotifera) in relation to temperature,salinity and food algae

Effects of temperature (18, 24, and 30°C), salinity (5–40 ppt, five intervals) and algal foods (Synechococcus sp., Chlorella pyrenoidosa, Isochrysis zhanjiangensis, Dunaliella salina and Tetraselmis cordiformis) on the life table demography of six geographical Brachionus plicatilis sensu stricto clones, which had been identified according to the prevalent taxonomy and biometric analysis of B. plicatilis sensu lato, were studied. The results showed that temperature, salinity and temperature × salinity significantly influenced the life history parameters. Genotype (clone) had no effect on the population growth rate but did influence the net reproductive rate, generation time and lifespan. All rotifer clones showed the expected increase in population growth rate with increasing temperature. B. plicatilis s. s. attained a higher population growth rate at low–medium salinities (5–20 ppt) than at high salinities (25–40 ppt). The equivalent spherical diameter (ESD) of food algae, salinity and ESD × salinity had significant effects on the life history parameters. In this case, genotype had no effect on population growth rate, net reproductive rate and generation time but did influence lifespan. The population growth rate of B. plicatilis s. s. evaluated against particle retention spectrum of algae at two salinities resulted in bell-shaped curves. Dunaliella salina with an ESD = 7.7 μm was considered to be the best food for B. plicatilis s. s. while Synechococcus appeared to be an inadequate food algae.