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.     

Preliminary comments on a redescription of Pseudolithophyllum fuegianum and Titanoderma conspectum (Rhodophyta,Corallinales) in Tierra del Fuego,Argentina

The effect of twelve drugs and chemical compounds on the narcosis of Brachionus plicatilis was studied using standardized laboratory conditions. Drug efficacy was compared by calculating EC₅₀ (effective concentration causing narcosis in 50% of animals), time necessary to reach narcosis in 50% of animals, concentration range of activity, and degree of extension after preservation. The local anesthetic Bupivacaine was found to be most effective by all criteria. Our previous data and preliminary field experiments indicated that drug sensitivity varies widely, even between congeneric taxa. The anesthetic effect of carbonated water was also investigated.     

Effect of salinity on competition between the rotifers Brachionus rotundiformis Tschugunoff and Hexarthra jenkinae (De Beauchamp) (Rotifera)

We studied the effect of different concentrations (0, 3, 6, 9 and 12 g l^–1) of sodium chloride at one food level of Chlorella (1×10⁶ cells ml^–1) on competition between the rotifers B. rotundiformis and H. jenkinae, both of which were isolated from a saline lake. The population growth experiments were conducted for 3 weeks. Both the rotifer species did not survive beyond one week at a salinity of 0 g l^–1. Regardless of salt concentration and the presence of a competitor, H. jenkinae reached higher densities than B. rotundiformis. When grown alone, both B. rotundiformis and H. jenkinae showed optimal peak population densities at the salinity of 6 and 9 g l^–1. Since biomass wise, B. rotundiformis was larger than H. jenkinae, it showed a lower numerical abundance. Thus, the maximum peak population densities of B. rotundiformis and H. jenkinae recorded in this study were 107±3 and 203±28 ind. ml^–1. The maximal rates of population increase for B. rotundiformis and H, jenkinae when grown alone were 0.264±0.003 and 0.274±0.004, respectively. Our results also indicated that B. rotundiformis and H. jenkinae coexisted better at a salinity of 6 and 9 g l^–1 of sodium chloride while a salinity of 3 g l^–1 favoured Hexarthra over B. rotundiformis. At 12 g l^–1, both the rotifer species grown alone or together showed lower growth rates compared to those at lower salinity levels. Except 0 g l^–1, in all other salinity treatments, H. jenkinae was a superior competitor to B. rotundiformis.     

Re-appearance of rotifers in hypersaline Mono Lake, California, during a period of rising lake levels and decreasing salinity

The surface elevation of Mono Lake, California, rose 2 m and mixed-layer salinities declined about 5 g kg^–1 during the 3 years (1995–1997) following the decision to restrict water diversions out of the Mono Basin. Abundant (18000 m^–2) Hexarthra jenkinae de Beauchamp were noted in pelagic samples in October 1997 after three decades of absence or very low abundance. Abundance subsequently increased to 100000 m^–2 in December 1997 before declining to low numbers through 1998 and 1999. The re-appearance of Branchionus plicatilis Müller in pelagic samples occurred in September 1998. B. plicatilis areal abundance increased to 15000 m^–2 in October–December of both 1998 and 1999 but was low throughout the rest of the year. Both rotifers were noted in nearshore ponds, but were only abundant in those with salinities below 53 g kg^–1. During 1998–1999 when the salinities of the upper water column were 73–75 g kg^–1, less saline shoreline habitats may have been seeding the offshore rotifer populations.     

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.     

Asexual and sexual reproduction in the rotifer Brachionus plicatilis cultured at different salinities

Two strains of the rotifer Brachionus plicatilis (L-type) differing in the levels of mictic female and male production, were grown in batch cultures with the alga Nannochloropsis gaditana as food, at two low (2.5 and 10), and two high (40 and 50) salinities. While both the low (strain S-1) and the high (strain S-3) sexual reproducing strains developed similar growth cycles at 2.5 and 10, the population growth response at 40 and 50 showed that; 1) in strain S-1, mixis can be suppressed in conditions that still allow asexual reproduction, and 2) in strain S-3 mictic female and male production are possible at nearly zero asexual population growth rates. In strain S-3, a double log linear relationship between the densities of males and females was found. These results show that mixis can occur over a wide ranges of female population density, and support the hypothesis that sexual reproduction is a strain dependent component of the general reproductive response.     

Influence of the concentration of oxygen on the swimming path of Brachionus plicatilis (Rotifera)

Brachionus plicatilis females were maintained for > 24 hours in water where the concentration of oxygen was precisely controlled (spherical flasks with 6 mg l^–1 or < 0.5 mg l^–1; food = dead Tetraselmis sp.). Each female was randomly taken from one flask and quickly placed in an observation chamber containing the same experimental conditions. The swimming path was videotaped (5 minutes); then the size of the female was measured. The tape was analyzed by automatic tracking (25 x,y coordinates of the center of the animal, in a 512 × 512 pixels space). The swimming path was analyzed for 45 females in both treatments. The speed (mm s^–1 body length^–1) was calculated for all trajectories, or only for those segments where females swam in a horizontal plane. This relative speed significantly decreased when the concentration of oxygen was very low. There was a negative correlation between the linear speed and the angular speed. The spatial sinuosity (S of Bovet & Benhamou, 1988) was calculated. The trajectories were significantly more sinuous when the concentration of oxygen was very low. These results could explain the accumulation of some rotifers in the oxycline; rotifers may spend more time in very low concentrations of oxygen by slowing and by turning more.     

Use of swimming speed and egg ratio as predictors of the status of rotifer cultures in aquaculture

This study evaluated the use of egg ratio (eggs rotifer^–1) and swimming speed (mm min^–1) as prediction criteria for production and culture quality in mass cultures of the rotifer Brachionus plicatilis. Egg ratio was determined to be a suitable predictor of rotifer growth and production in the cultures. Low egg ratios (i.e., 0–0.17 eggs rotifer^–1) indicate reduced rotifer population over time (i.e., negative net population growth rates). However, at this time egg ratio dynamics are not suitably understood to predict in advance a sudden population collapse.Swimming speed of reproductive, egg-carrying females in the exponential growth phase was 40–45 mm min^–1. During exponential growth swimming speed was independent of the food used. Lower swimming speeds were obtained in late stationary phase (10–25 mm min^–1) when yeast was used as a food source. Both environmental factors (e.g., accumulating metabolites) and changes in nutritional state of the rotifers may have affected the swimming speed, but environmental factors appear to be the most important. We believe that swimming speed has the potential of becoming an accurate predictor of culture quality in mass cultures of rotifers.     

Removal of Surface Glycoproteins and Transfer among <Emphasis Type="Italic">Brachionus</Emphasis> species

Glycoproteins on the body surface of females of the rotifer Brachionus plicatilis are a key signal in their mate recognition system. When B. plicatilis Russian strain females were exposed to 50 mM EDTA or EGTA, several surface glycoproteins were removed. Females exposed to EDTA died, but remained intact and were used in mating bioassays with conspecifics males. Live control females elicited a male mating response in 21% of encounters, freeze-killed control females elicited responses in 23%, but EDTA extracted females elicited a mating response in only 5% of encounters. At least some of the EDTA-extractable proteins on the surface of females appear to be critical to male mate recognition. EDTA treated females could be exposed to proteins extracted from other females and some proteins re-attached to their body surface, restoring their attractiveness to males. SDS-PAGE of these proteins revealed 15–17 prominent bands, most ranging in molecular mass from 66 to 12 kD. The EDTA-extractable proteins were separated using ion exchange chromatography and each fraction was tested for its ability to restore female attractiveness. When proteins in fraction 22 were bound to females, they restored 80% of the females’ ability to elicit male mating responses. Exposing EDTA treated females to bovine serum albumin or casein had no effect on their attractiveness to males. EDTA treated females from different Brachionus clades and species were exposed to proteins from fraction 22. Female attractiveness could be restored in most clades of B. plicatilis, but no transfer of mating attractiveness was observed to B. rotundiformis or B. ibericus females. Conspecific males treated with EDTA and exposed to proteins in fraction 22 could not be feminized and made attractive to other males. A sexual dimorphism in surface proteins therefore exists between B. plicatilis females and males. Successful transfer of glycoproteins critical in mate recognition is dependent on signal glycoprotein structure and the structure of the other proteins present on the surface of females.     

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.     

Nomenclatural consequences of some recent studies on Brachionus plicatilis (Rotifera,Brachionidae)

Recent studies on morphology, karyotype, genetics including allozyme constitution and reproductive behaviour of S- and L-type B. plicatilis showed that these types are best treated as different species. A reexamination of existing available names revealed B. plicatilis O. F. Müller, 1786 and B. rotundiformis Tschugunoff, 1921 as the correct names for the L- and S-type, respectively.     

Observations on the susceptibility of some protists and rotifers to predation by asplanchna girodi

Direct observations of behavioral interactions show that the predator A. girodi: 1) easily ingested Synchaeta pectinata, two forms of Keratella cochlearis cochlearis, and individuals of Conochilus unicornis and C. dossuarius enzymatically dissociated from their matrix; 2) rarely, if ever, captured Kellicottia bostoniensis and intact Conochilus; and 3) generally rejected the peritrich ciliate Rhabdostyla sp. and the dinoflagellate Peridinium sp. Coloniality and secretion of a gelatinous matrix in Conochilus can be viewed as adaptations to limit mortality from invertebrate predation. Intraspecific variability in the feeding responses of A. girodi is considered.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

An algal medium based on fertilizers and its evaluation in mariculture

Five agricultural fertilizers were tested as potential nutrient enrichments for the mass culture ofTetraselmis suecica. Maximum algal growth was observed for the trade fertilizer Igromurtonik^R (Murphy Ltd, England) adjusted for a nitrate to phosphate ratio of 24:1. The gross biochemical composition ofT. suecica grown in the enriched fertilizer was compared to the composition of the alga grown in control medium. The nutritional value of the algal material was then tested on the rotiferBrachionus plicatilis. The medium based on the fertilizer is as an inexpensive substitute for mass algal culture ofTetraselmis suecica, a food source for the rotiferBrachionus plicatilis.     

Interrelations of rotifers with predatory and herbivorous Cladocera: a review of Russian works

Publications in Russian on the influence of epibiontic rotifers on cladocerans as well as on predator-prey interrelations between cladocerans and rotifers are reviewed. Proales daphnicola and Brachionus rubens are common epibionts of Cladocera. The biology of these species is described including the choice of host, feeding, reproduction and impact on the host. Representatives of the families Daphniidae and Moinidae are most readily colonized. At high densities of epibiontic rotifers, a high percentage of young Cladocera die. Predators consuming cladocerans (mainly Bosminidae) belong to the family Asplanchnidae. Rotifers are consumed by the predatory cladocerans Leptodora and Polyphemus. The results of functional response experiments by the author with P. pediculus feeding on Synchaeta pectinata, Asplanchna priodonta, colonial and solitary Conochilus unicornis, and Platyias patulus are given. In the range of prey densities of 100–1600 1^–1 a functional response was found in all the rotifers except P. patulus. Colonies of C. unicornis were not consumed. The highest level of feeding rate saturation was observed in Synchaeta pectinata. Various adaptations in prey morphology prevented effective predation: coloniality, large size of the prey and hard lorica with spines.     

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.     

Amphora margalefii Tomàs var. lacustris P. Sánchez var. nova,a new brackish water diatom

The rotifer Brachionus plicatilis can grow in a wide range of salinities and temperatures, but rapid shifts in both salinity and temperature may result in immobilized, non-swimming rotifers. The goal of this study was to examine the effect of perturbations in temperature and salinity on the swimming pattern of the rotifer.Only slight changes in mobility were observed when rotifers were exposed to changes in temperature (from 20 °C to 8–30 °C) and to an increase in salinity (from 20% to 30%). When the salinity was reduced to 15% and 5%, the proportion of mobile rotifers was reduced to 50% and 5%, respectively. The rotifers were throughout more resistant to perturbations in temperature than to those of salinity.Combined temperature and salinity perturbations compared to perturbations in each factor separately suggested a synergetic effect of temperature and salinity on the rotifers locomotion. Transfer from cultivation conditions to low salinity (5%) and high temperature (28 °C) resulted in very low percent of mobile rotifers (0–10%). However, if the temperature was reduced to 8 °C concomitant with the changes in salinity, the percent of mobile rotifers was 85%.Rotifers use a high share of their metabolic energy for locomotion, and it is therefore not surprising that perturbations in salinity and temperature may result in partial or complete immobilization.     

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.     

An individual-based population model for the prediction of rotifer population dynamics and resting egg production

Most species of rotifers have a combination of sexual and asexual reproduction, with sexual reproduction resulting in resting eggs, which can lay dormant for long periods. The occurrence of sexual reproduction affects population dynamics through the temporary presence of male rotifers, and a reduction in the growth of the number of female rotifers. A previously published, individual-based model used dynamic energy budget theory to describe rotifer food intake, growth, egg production, and mortality, but assumed asexual reproduction only. In the current study, we have expanded the model to describe the entire reproductive cycle of the rotifers, making it usable for investigating relationships, such as those between the signal triggering mictic egg production, and the timing and number of resting eggs produced. The model is intended for use in predicting the specific future development of cultures, for instance, as a process model in rotifer or resting egg production for aquaculture. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

Comparative population dynamics of the rotifers Brachionus angularis and Keratella cochlearis

The results indicate that the two rotifer species Brachionus angularis and Keratella cochlearis have different life history strategies. Brachionus angularis appeared to be an r-strategist, showing higher reproductive, mortality and population growth rates than K. cochlearis. Additional evidence is offered suggesting that K. cochlearis is a K-strategist. Although K. cochlearis had a lower growth rate than B. angularis, this was compensated by a lower mortality rate, especially in pre-reproductive individuals. In addition, K. cochlearis allocated energy to reproduction more efficiently than B. angularis.