Mating behavior and mate recognition pheromone blocking of male receptors in Brachionus plicatilis Müller (Rotifera)

Copulatory behavior of three S and three L type B. plicatilis strains from different geographic areas was analyzed. A 29 KD surface glycoprotein on females, characterized as a Mate Recognition Pheromone (MRP), binds to receptors in the corona of males and blocks mate recognition. Blocking was observed in all S and L strains even though the MRP was isolated from a single L-type strain. Binding was quantified using image analysis and a 20-fold difference was observed among strains. A direct relationship between the male discrimination of females and the intensity of MRP binding to male receptors was found. This relationship might be useful as a tool to examine variation in the mate recognition systems of other rotifer species.     

Competition between Brachionus calyciflorus Pallas and Brachionus patulus (Müller) (Rotifera) in relation to algal food concentration and initial population density

Competitive laboratory experiments between Brachionus calyciflorus and B. patulus were conducted at low (1×10⁶ cells ml^–1) and high (3×10⁶ cells ml^–1) densities of Chlorella vulgaris and four initial inoculation densities (numerically, 100% B. calyciflorus; 75% B. calyciflorus + 25% B. patulus; 50% each of the two species; 25% B. calyciflorus + 75% B. patulus and 100% B. patulus). Population densities were enumerated and the medium was changed daily for 20 days. B. patulus was a superior competitor in low food density regardless of inoculation density. At high food density, B. calyciflorus showed higher population growth in the first week but thereafter was outcompeted by B. patulus regardless of initial density. When grown alone, B. calyciflorus reached peak abundances (mean ± standard error) of 31±3 and 81±7 individuals ml^–1 at low and high food densities, respectively. The corresponding values for B. patulus were 130±2 and 306±13. The adverse effects of B. patulus on the peak abundances of B. calyciflorus were higher at low food concentration. Data on egg ratios (eggs female^–1) revealed an inverse relation with population abundance of both tested rotifer species. Our results indicated that the rate of population increase of a species was not a good indicator of its competitive ability. Instead, the ability to reproduce under continuously diminishing food resources (until a threshold level) was responsible for the competitive edge of B. patulus over B. calyciflorus. This was further influenced by the relative inoculation densities of the tested rotifer species and the offered food densities.     

Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Mictic patterns of the rotifer Brachionus plicatilis Müller in small ponds

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes (S and L). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study S and L morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.     

Effect of periodical starvation on the life history of Brachionus plicatilis O.F. Müller (Rotifera): a possible strategy for population stability

To estimate the changes in the life history of the rotifer Brachionus plicatilis O.F. Müller under starvation, we carried out an individual culture and determined the effects of periodical food deprivation on its asexual reproductive characteristics such as lifespan, reproductive period, age at first egg and offspring production, and lifetime fecundity (total number of offspring produced in her lifetime). Rotifers were fed for 1-3 h daily, and were then starved until the next day. Control animals were fed throughout their lifespan. Starved rotifers matured and produced their first offspring at an older age than the control animals. The periodical starvation resulted in a decrease in the lifetime fecundity to less than half that of the non-starved control. The reproductive period and lifespan were 2-3 times longer in the starved animals than in the control animals. The negative relationship between lifespan and lifetime fecundity is interpreted as a trade-off in an alternative life-history strategy of rotifers under starved conditions. The great decrease in fecundity and extension of lifespan enables rotifers to compensate to keep the population in equilibrium.     

Diurnal vertical distribution of rotifers (Rotifera) in the Chara zone of Budzyńskie Lake,Poland

Diurnal vertical distribution of rotifers was investigated in the Chara bed and the water immediately above it in the shallow region (ca. 1 m depth) of Budzyskie Lake (Wielkopolski National Park, Poland) in early September 1998. Eighty one rotifer species were identified – 71 among Chara and 59 in the open water. Significant differences in rotifer densities were observed in the Chara, with highest numbers during the day (2316 ind. l^–1) and lowest numbers early morning (521 ind. l^–1) and at dusk (610 ind. l^–1). Above the Chara, the numbers of rotifers did not change significantly (615–956 ind. l^–1). Littoral- or limnetic-forms differed in their diel vertical distribution between both zones. One group of littoral species was characterized by increased densities in the Chara in the daytime, while a second group increased in density during the night. The densities of limnetic species, which were much higher in open water, decreased in the morning or daytime in this zone. These differences in the diel behaviour of particular groups of rotifers may be dependent on microhabitat and may also be related to different kinds of predation, the exploitative competition for shared food resources between rotifers and crustaceans, as well as typical adaptation to littoral or limnetic life.     

Dependence of temperature on loss rates of rotifers,lipids, and ω3 fatty acids in starved Brachionus plicatilis cultures

Rotifer cultures of Brachionus plicatilis (SINTEF-strain, length 250 m) rich in 3 fatty acids were starved for > 5 days at variable temperature (0–18 °C). The net specific loss rate of rotifer numbers were 0.04 day^–1 (range 0–0.08 day^–1) at 5–18 °C, but reached values up to 0.25 day^–1 at 0–3 °C. The loss rate was independent on culture density (range 40–1000 ind ml^–1), but was to some extent dependent on the initial physiological state of the rotifers (i.e., egg ratio).The loss rate of lipids was 0.02–0.05 day^–1 below 10 °C, where the potential growth rate of the rotifer is low (0–0.09 day^–1). The loss rate of lipids increased rapidly for higher temperatures where the rotifer can maintain positive growth, and reached 0.19 day^–1 at 18 °C. The Q₁₀ for the lipid loss rate versus temperature was higher than the Q₁₀ for respiration found in other strains. This may suggest that other processes than respiration were involved in lipid catabolism. The content of 3 fatty acids became reduced somewhat faster than the lipids (i.e. in particular 22:6 3), but the fatty acid per cent distribution remained remarkably unaffected by the temperature during starvation.The results showed that rotifer cultures could be starved for up to 4 days at 5–8 °C without essential quantitative losses of lipids, 3 fatty acids, and rotifers. The rotifers exhausted their endogenous lipids through reproduction (anabolism) and respiration (including enhanced locomotion) at higher temperatures. At lower temperatures, the mortality rate became very high.     

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.     

Speciation of dimethyltin(IV) – and trimethyltin(IV) – carbocysteinate and – glutamate systems in aqueous media

Abstract

The formation of complex species in the dimethyltin(IV) and trimethyltin(IV)-carboxymethyl-L-cysteinate (carbocysteinate) systems in NaCl_aq, at different ionic strengths, and in a multicomponent Na⁺, K⁺, Ca²⁺ ,Mg²⁺, Cl^? and SO₄^2-? medium representative of the seawater major composition, is discussed. Experimental results give evidence for the formation of the following species (L = carbocysteinate): [(CH₃)₂Sn(L)]⁰, [(CH₃)₂Sn(HL)]⁺, [(CH₃)₂Sn(OH)(L)]^?, [(CH₃)₂Sn(OH)₂(L)]^2? in the DMT–CCYS system, and [(CH₃)₃Sn(HL)]⁰, [(CH₃)₃Sn(L)]^? and [(CH₃)₃Sn(OH)(L)]^2? in the TMT-CCYS system. The ionic strength dependence of formation constants was taken into account by an extended Debye Hückel type equation and by the SIT (Specific ion Interaction Theory). Measurements were carried out also on the dimethyltin(IV)-glutamate and trimethyltin(IV)-glutamate systems in NaCl_aq, owing the strict similarity of glutamate and carbocysteinate. Results obtained show the formation of complex species having the same stoichiometry as those formed in the DMT- and TMT-carbocysteinate systems, with very similar stability, confirming that carbocysteinate behaves as a dicarboxylic amino acid without involving the sulfur-bridge potential binding site in metal coordination.     

Effect of diets containing γ-linolenic acid on n-6 highly unsaturated fatty acid content of rotifer (Brachionus plicatilis)

The effect of various diets containing linoleic and/or -linolenic acids was studied on n-6 fatty acid composition of the rotifer Brachionus plicatilis. The rotifer's abilities for transformations of n-6 fatty acids were evaluated. Diets containing only linolenic acid as n-6 fatty acid induced low levels of other n-6 fatty acids in rotifers while a diet containing also -linolenic acid led to substantial amounts of di homo -linolenic acid in the rotifers through elongation. Desaturation of -linoleic acid to gamma linolenic appears to be the limiting factor of n-6 highly unsaturated fatty acid biosynthesis by the rotifer. Two sets of experiments were compared using different techniques and different sources of -linolenic acid: Spirulina in inert food or borage oil in emulsion with baker's yeast. Rotifers fed with inert diet with Spirulina contained arachidonic acid while those fed with borage oil had very low arachidonic content. High level of n-3 fatty acids incorporated into the diets seemed to exert inhibitory effects on n-6 transformation rate.     

Horizontal distribution of cladocerans in arctic Greenland lakes – impact of macrophytes and fish

Submerged macrophytes may play an important role as a refuge for zooplankton against predators. However, a recent study suggests that their importance depends on the trophic state of the lake. We studied the impact of fish and macrophytes on the horizontal distribution of pelagic cladocerans in 56 oligotrophic arctic Greenland lakes. In north-east and western Greenland, zooplankton was sampled in the near-shore (littoral) and central (pelagial) part of all lakes and fish were sampled with multiple mesh-sized gill nets. Macrophytes were visually estimated in the littoral. In north-east Greenland, 5 taxa of cladocerans were found, while 14 taxa were recorded in western Greenland. Daphnia pulex occurred only in fishless lakes in both northeast and western Greenland and avoided the near-shore areas in the shallow and deep lakes. Bosmina spp. and Holopedium gibberum were evenly distributed between the littoral and the pelagial in the deep and shallow fishless lakes. However, their near-shore density was lowest in the presence of fish. Macrophyte-related and benthic cladocerans concentrated either in the littoral or were evenly distributed between the littoral and the pelagial, irrespective of depth and fish presence or absence. Macrophytes had no impact on the horizontal distribution of pelagic cladocerans. Thus, it is concluded that horizontal heterogeneity of Bosmina spp. and Holopedium gibberum might be affected by the presence of fish.     

Influence of the age of algae fed to rotifers (Brachionus plicatilis O.F. Müller) on the expression of mixis in their progenies

Summary The sequence of the appearance of mixis in the rotifer Brachionus plicatilis was followed among the descendents of amictic rotifers transferred from a high salinity media (40 S) to a low one (9 S). All the neonates that hatched from the amictic eggs, after being transferred to a low salinity, were amictic. Each one of these neonates was cultured individually and its offspring removed periodically every 8–10 h. It was observed that throughout their reproductive phase, these parental females retained their potential to produce either mictic or amictic offspring. All the first produced neonates developed into amictic females, but among those produced later, three patterns were prevalent. The prevalent pattern (type A) was one in which the probability of a neonate being mictic increased towards the middle of the parents' reproductive phase and was followed by a slow decline. In the second pattern (type B), the probability of a daughter being mictic was constant throughout the parents' reproductive phase. It is suspected that the quality of food supplied to the rotifers determines the appearance of patterns, A, B or C. It is postulated that the innate capacity of rotifers to undergo mixis is genetically controlled, while its expression is modulated by environmental conditions.     

Resting eggs of the marine rotifer Brachionus plicatilis Müller: development,and effect of irradiation on hatching

Diapause and hatching of Brachionus plicatilis Müller resting eggs were examined through histological and optical approaches. Compound microscope observations on 1% toluidine blue-stained embryo sections suggests that the total number of nuclei in an embryo during the internal diapause period increased from 22 on Day 2 to 39 (each n = 1) on Day 6. The outer layer of embryo membrane gradually thickens from 1.2 (Day 0) to 4.0 µm (Day 8) (each n = 10).Resting eggs that have completed maturation and are in the external diapause period require light for hatching. The threshold of light (halogen lamp) intensity for hatching was estimated to be 4400 lux for 30 min. Hatching rate decreased with longer wavelength irradiation (mercury lamp). Irradiation at more than 350 nm caused 1–25% hatching, but it reached 50–60% at 250–310 nm light. The addition of hydrogen peroxide or prostaglandins (E ₁, E ₂ or F ₂) caused resting egg hatching even in darkness. The production of peroxide in seawater caused by light as well as the oxidation of fatty acid to prostaglandins inside the embryo is a possible mechanism of resting egg hatching.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

Biochemical properties and mechanism of action of a vanadyl(IV) – aspirin complex on bone cell lines in culture

A recently synthesized vanadyl(IV) complex with aspirin[VO(aspirin)ClH₂O]₂, has been thoroughly investigated by physicochemical techniques. In order to support the proposed structure, stoichiometry and the coordination sphere of the vanadium center, some studies such as elemental analysis, electronic (diffuse reflectance) and vibrational (infrared) spectroscopies, magnetic susceptibility, as well as the thermal behavior, were carried out. The bioactivity of the vanadium complex (VOAspi) was evaluated on two osteoblast-like cell lines in culture, being its cytotoxic effects stronger than the vanadyl cation as assessed by morphological changes and lipid peroxidation. These effects may be partially explained through the induction of the expression of Erks (Extracellular signal-regulated kinases) and the inhibition of the PTPases (Phosphotyrosine phosphatases) present in the cellular extracts.     

Patching of gangliosideM1 in human erythrocytes – distribution of CD47 and CD59 in patched and curved membrane

Membrane rafts may act as platforms for membrane protein signalling. Rafts have also been implicated in the sorting of membrane components during membrane budding. We have studied by fluorescence microscopy cross-linking of ganglioside GM1 in the human erythrocyte membrane, and how membrane proteins CD47 and CD59 distribute in GM1 patched discoid cells and calcium-induced echinocytic cells. Patching of ganglioside_M1 (GM1) by cholera toxin subunit B (CTB) plus anti-CTB resulted in the formation of usually 40–60 GM1 patches distributed over the membrane in discoid erythrocytes. Pre-treatment of erythrocytes with methyl-β-cyclodextrin abolished GM1 patching. GM1 patching was insensitive to pre-fixation (paraformaldehyde) of cells. Patching of GM1 did not affect the discoid shape of erythrocytes. Membrane proteins CD47 and CD59 did not accumulate into GM1 patches. No capping of patches occurred. GM1 accumulated in calcium-induced echinocytic spiculae. Also CD59, but not CD47, accumulated in spiculae. However, CD59 showed a low degree of co-localization with GM1 and frequently accumulated in different spiculae than GM1. In conclusion, our study describes a novel method for examining properties and composition of rafts. The study characterizes raft patching in the human erythrocyte membrane and emphasizes the mobility and ‘echinophilicity’ of GM1. Glycosyl phosphatidylinositol-anchored CD59 was identified as a mobile ‘echinophilic’ but ‘raftophobic_GM1’ protein. Largely immobile CD47 showed no segregation.     

Influences of geometrical and mechanical properties of bone tissues in mandible behaviour – experimental and numerical predictions

The properties and geometry of bone in the mandible play a key role in mandible behaviour during a person’s lifetime, and attention needs to be paid to the influence of bone properties. We analysed the effect of bone geometry, size and bone properties in mandible behaviour, experimenting on cadaveric mandibles and FE models. The study was developed using the geometry of a cadaveric mandible without teeth. Three models of cadaveric condyles were experimentally tested with instrumented with four rosettes, and a condyle reaction of 300 N. Four finite element models were considered to validate the experiments and analyse mandible behaviour. One numeric model was simulated with 10 muscles in a quasi-static condition. The experimental results present different condyle stiffness’s, of 448, 215 and 254 N/mm. The values presented in the rosettes are influenced by bone geometry and bone thickness; maximum value was ?600 με in rosette #4, and the maximum strain difference between mandibles was 111%. The numerical results show that bone density decreases and strain distribution increases in the thinner mandible regions. Nevertheless, the global behaviour of the structure remains similar, but presents different strain magnitudes. The study shows the need to take into account bone characteristics and their evolutions in order to improve implant design and fixation throughout the patient life. The change in bone stiffness promotes a change in maximum strain distribution with same global behaviour.     

Relationship between the distribution of euphausiids and baleen whales in the Antarctic (35°E – 145°W)

Simultaneous whale sighting and hydroacoustic surveys were conducted from a research vessel in the Antarctic to examine the relationship between the distribution of euphausiids and baleen whales. High densities of minke whales and large aggregations of euphausiids were observed along the ice edge over the continental slope in the southeast region of area IV and in the southwest region of area V. The results suggest that the continental slope zone that coincides with the ice edge would be an important minke whale feeding area. Minke whales were rarely sighted in the offshore region even if euphausiids were abundant. Distributions of humpback whales were correlated with high euphausiid density zones, regardless of the bottom topographic features. Several groups of blue whales were sighted in the small area along the ice edge where euphausiids were abundant, but sightings were too few to draw any general conclusion about the relationship between blue whales and euphausiids. Both baleen whales and euphausiids were scarce in the area east of 170°W where sea ice covered the continental shelf and slope zone.