Salinity dependence of sexual and asexual reproduction in the rotifer Brachionus plicatilis

A salinity dependent mictic response was observed in a clone of Brachionus plicatilis cultured in the 2 to 4 salinity range. This response was related to asexual exponential reproduction rates (G) and could be divided into three categories: (a) no mixis occurred at a salinity of 35 S and above, where G values were lower than 0.30 d^-1, (b) low mictic levels in rotifers cultured at 2 and 30 S, where G values ranged between 0.40 to 0.50 d^-1, and (c) high mictic levels in rotifers cultured at salinities ranging between 4 and 20 S, where G values ranged between 0.50 to 0.85 d^-1. Fluctuations in mictic levels varied with time during the course of the experiments. Results suggest that salinity conditions leading to optimal parthenogenic reproduction also support mixis.     

Food selectivity of the rotifer Brachionus plicatilis feeding on phytoplankton

Food selectivity was examined in amictic female rotifers, Brachionus plicatilis, fed Chlamydomonas sp. and Olisthodiscus sp. Filtering and ingestion rates of the rotifers in a single food suspension were higher with Chlamydomonas sp. than with Olisthodiscus sp. In every mixture of two food species, the apparent filtering rate on Chlamydomonas sp. was much higher than that on Olisthodiscus sp. Total filtering rate and apparent filtering rate in food suspensions containing Chlamydomonas sp. decreased with increasing cell concentration of Chlamydomonas sp. Total and apparent filtering rates were calculated from the decrease in total food concentration as well as from decreases in concentrations of each of the two food algae, respectively. B. plicatilis ingested Olisthodiscus sp. at an extremely low constant rate in all mixtures. The degree of food selectivity of the rotifers fed Chlamydomonas sp. (i.e., selective filtration) from the mixture of two food algae decreased with increasing cell concentration of Chlamydomonas sp. Filtering and ingestion rates of rotifers fed senescent Chlamydomonas sp. were relatively lower than those fed Chlamydomonas sp. in the exponential phase. This indicates that Brachionus plicatilis displays selectivity in regard to condition of cells as well as type of food.     

How does salinity tolerance influence the distributions of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> sibling species?

Environmental salinity is important in defining Brachionus plicatilis sibling species distributions. However, while salinity influences distributions, sibling species often co-exist. Three different mechanisms potentially account for the partial co-occurrence of sibling species: (1) siblings have differing salinity tolerances that partially overlap; (2) siblings physiological tolerances may be commonly broad, but relatively small differences in tolerances differentiate distributions via interactions e.g. competition; or (3) siblings distributions may be influenced by physical factors other than salinity. Here, we assess the extent of salinity tolerance in three B. plicatilis sibling species (B. plicatilis 6TUR, B. plicatilis IOM and B. rotundiformis 6TOS) by measuring population growth rate (μ, day⁻¹) and egg development time in response to salinity (5–60‰) and salinity fluctuations (≤ Δ40‰). Sibling species were identified by analysis of the mitochondrial COI gene, and salinity responses were compared by regression analysis. Responses differed significantly between siblings, although the broad trends were similar. Positive growth occurred at all salinities, and highest growth rates ranged between 0.93 and 1.08 day⁻¹ at 16–18‰. Rapid changes in salinity reduced growth rates, but net mortality occurred only in one treatment (100% mortality on transfer from 10 to 40‰). Egg development time was largely invariant with salinity except for B. plicatilis IOM and where rotifers were transferred from 30 to 60‰. We indicate that several siblings are similarly euryhaline and tolerate salinity fluctuations. Undoubtedly, wide tolerances in B. plicatilis are adaptations to ephemeral and seasonally variable habitats. Given common broad salinity tolerances, it is unlikely that the differential distributions of sibling species are a direct result of physiological constraints. Instead, we illustrate using a simple model that subtle differences in physiological tolerances may have important impacts on interactions between sibling species, which may in turn influence distributions.     

Identification of a sex pheromone from a rotifer

The first isolation and characterization of a sex pheromone from a zooplankter is reported. A 29 kdalton glycoprotein on the surface of females of the marine rotifer Brachionus plicatilis acts as a contact-mating pheromone. This glycoprotein (gp29) is glycosylated with oligosaccharides containing Nacetylglucosamine, mannose, and fucose residues, and these oligosaccharides are necessary for male recognition of females. Males detect this signal by contact chemoreception with receptors located in their corona. Binding of purified glycoprotein to male receptors reduces mating attempts by 93%. An antibody to the glycoprotein binds to females, reducing male mating attempts by 86%. When purified gp29 is bound to sepharose beads, it is sufficient to elicit male mating behavior. This glycoprotein is likely to play a key role in the evolution and maintenance of reproductive isolation in rotifers.     

Effects on larval crabs of exposure to algal toxin via ingestion of heterotrophic prey

We tested whether ingesting toxic algae by heterotrophic prey affected their nutritional value to crab larval predators, using toxic algal strains that are either ingested directly by larval crabs or rejected by them. Ingestion of toxic strains of the dinoflagellates Alexandrium andersoni and A. fundyense by the rotifer Brachionus plicatilis was confirmed. Rotifers having ingested either algal type for five days were fed to freshly hatched larvae of three crab species, with larval survival and stage durations determined. For both algal/rotifer treatments in all three crab species, larvae fed algae directly died during the first zoeal stage, while those fed rotifers that had been fed either algal strain survived to the experiment’s end (zoeal stage 3). Survival was lower, and stage duration longer, for larvae fed rotifers cultured on toxic algae when compared to those fed non-toxic algae. The role of toxic algae in the planktonic food web may be influenced by its direct or indirect ingestion by larval crabs.     

Mass culture of the rotifer Brachionus plicatilis and its evaluation as a food for larval anchovies

Growth rates of anchovy larvae, Engraulis mordax, reared for 19 days under constant environmental conditions on a diet of laboratory-cultured organisms, exceeded the growth rates of anchovies fed on a diet of wild plankton. The rotifer Brachionus plicatilis was found to be a nutritous food source when fed to the larvae in concentrations of 10 to 20/ml and in combination with the dinoflagellate Gymnodinium splendens (100/ml). Optimum conditions were determined for mass culture of the rotifer. A high food concentration was the most important parameter needed to assure a high yield of rotifers. Large volumes (464 I) of the unicellular flagellate Dunaliella sp. were cultured for feeding the rotifers. The rotifer culture technique described produces approximately 2.5×10⁶ organisms/day, providing a reliable food source for rearing studies. The lengths of B. plicatilis (without eggs) ranged between 99 and 281 , most rotifers being larger than 164 and less than 231 . Individuals weighed 0.16 g and contained 8×10^-4 cal.     

Changes in life histories of cladocerans (Cladocera) from the rotifer-mediated allelochemicals

ABSTRACT

We hypothesised that at higher densities herbivorous rotifers through their allelochemicals affect the survival and reproduction of cladocerans. To test this, we separately cultured three rotifers (Brachionus calyciflorus, Brachionus havanaensis, and Plationus patulus) and three cladocerans (Ceriodaphnia dubia, Daphnia pulex, and Moina macrocopa). Chlorella vulgaris was used as food and reconstituted moderately hard water was used as medium. The conditioned-medium (CM) obtained from each of rotifer species was used to conduct cohort life table tests of cladocerans. SDS PAGE electrophoresis revealed the molecular weights of proteins present in the rotifer conditioned medium were 55–63?kDa. Compared to C. dubia and D. pulex, Moina was more resistant to rotifer-mediated chemicals. Gross reproductive rate of C. dubia was significantly stimulated by the rotifer-CM. However, gross and net reproductive rates of D. pulex were strongly reduced (72–85%) due to P. patulus-CM but not from the other two rotifer species. The rate of population increase of M. macrocopa was significantly reduced by the rotifer CM. Overall, about 46% (adverse effect: 24%; stimulatory impact: 22%) of the life history variables in controls significantly differed from rotifer-CM treatments, thus proving our hypothesis that rotifer-mediated allelochemicals had some effect on the cladoceran life history variables.     

The trophic linkage between zooplankton and benthic suspension feeders: direct evidence from analyses of bivalve faecal pellets

Using radiotracer (¹⁴C) and microscopic observation, we demonstrated that mussels (Mytilus edulis and Perna viridis) could be predators of mesozooplankton (rotifer Brachionus plicatilis). At radio-labelled rotifer densities of 0.1, 0.2, 0.5, 1.0 individual ml⁻¹, faecal pellets of mussels showed different degrees of radio signals and most of the faecal pellets were expelled 4 h after pulse feeding on rotifers. The maximum gut retention time (GRT) of ¹⁴C-labelled rotifers in the digestive diverticula did not o show any significant difference between the two mussel species or the different densities of rotifers, and the averaged GRT was 43.4±3.06 h (mean ± SE). At a rotifer density of 4.5 individual ml⁻¹, rotifer lorica pieces and rotifer bodies without eggs were found in faeces of M. edulis, while in the pseudofaeces, only complete rotifer bodies were found.     

Prey selection by larval weakfish (Cynoscion regalis): the effects of prey size,speed, and abundance

We examined feeding by larval weakfish, Cynoscion regalis (Bloch and Schneider), in laboratory experiments conducted during the 1991 spawning season. under natural conditions weakfish larval development is ca. 3 wk, and we ran separate experiments with larvae of five different ages (5, 8, 11, 14, and 17 d post-hatching). We used two different size classes of rotifers (Brachionus plicatilis) and brine shrimp nauplii (Artemia sp.) as prey organisms. Contrary to results of previous research, weakfish larvae did not select prey based on size alone. When prey abundance was above 100 itemsl^-1 weakfish, larvae always chose large rotifers (length = 216 m) over small rotifers (length = 160 m). At 11 d post-hatching, larvae switched their diet from large rotifers to small brine shrimp nauplii (length = 449 m); however, when fed small rotifers and small brine shrimp nauplii the change in diet occurred at 14 d post-hatching. This pattern of selectivity was maintained in each larval age class. Early-stage larvae (5 and 8 d post-hatching) did not feed selectively when prey abundance was less than 100 itemsl^-1. Late-stage larvae (17 d post-hatching) fed selectively at abundances ranging from 10 to 10000 items^-1. Lwimming speeds of prey items, which ranged from 1 to 6 mms^-1, had no consistent effect on prey selection. These results suggest that weakfish larvae are able to feed selectively, that selectivity changes as larvae age, and that selectivity is also influenced by prey abundance.     

Two different proteases produced by a deep-sea psychrotrophic bacterial strain,<Emphasis Type="Italic"> Pseudoaltermonas</Emphasis> sp. SM9913

A psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913, was isolated from deep-sea sediment collected at 1,855 m depth. Two proteases produced by Pseudoaltermonas sp. SM9913 were purified, MPC-01 and MCP-02. MCP-01 is a serine protease with a molecular weight of 60.7 kDa. It is cold-adapted with an optimum temperature of 30–35°C. Its K_m and E_a for the hydrolysis of casein were 0.18% and 39.1 kJ mol^–1, respectively. It had low thermostability, and its activity was reduced by 73% after incubation at 40°C for 10 min. MCP-02 is a mesophilic metalloprotease with a molecular weight of 36 kDa. Its optimum temperature for the hydrolysis of casein was 50–55°C. The K_m and E_a of MCP-02 for the hydrolysis of casein were 0.36% and 59.3 kJ mol^–1, respectively. MCP-02 had high thermostability, and its activity was reduced by only 30.5% after incubation at 60°C for 10 min. At low temperatures, Pseudoaltermonas sp. SM9913 mainly produced the psychrophilic protease MCP-01.Communicated by O. Kinne, Oldendorf/Luhe     

The digestive protease,chymotrypsin, as an indicator of nutritional condition in larval red drum (Sciaenops ocellatus)

Laboratory-reared red drum (Sciaenops ocellatus) larvae were used to evaluate the potential of chymotrypsin as an indicator of nutritional condition in marine fish larvae. The response of chymotrypsin activity to food deprivation and reductions in nutrient intake was determined. Enzyme activity declined rapidly to undetectable levels in food-deprived larvae 6–18 days old. Larvae fed poor-quality live prey (starved rotifers, Brachionus plicatilis) exhibited reductions in growth (18%) and enzyme activity (84%) relative to larvae fed high-quality prey (enriched rotifers). Potential sources of variation in chymotrypsin activity unrelated to nutritional status, including diel periodicity, and exogenous enzymes sources were examined. A diel pattern in chymotrypsin activity was detected with an 8.7-fold increase in activity occurring from low to high points during a 24-h period. Highest activity levels occurred late in the day (1600 hours) and lowest activity in the morning prior to feeding (0800 hours). The estimated contribution of exogenous enzymes from prey in the digestive tract to measurements of larval enzyme activity was small, reaching a maximum of 4.1% on day 18 in well-fed larvae. Results indicate that exogenous enzymes will not lead to the misclassification of larvae in poor condition. A relationship between chymotrypsin activity and standard length was established for well-fed and food-deprived larvae that could potentially be used to determine the nutritional condition of wild-caught larvae.     

Transfer of the chlorinated hydrocarbon PCB in a laboratory marine food chain

The transfer of chlorinated hydrocarbons (CHC) in a laboratory simulation of a three trophic level marine food chain was studied. The food chain consisted of the algal flagellate Dunaliella sp., the rotifer Brachionus plicatilis, and the larva of the Northern anchovy Engraulis mordax. CHC were introduced into the seawater at concentrations representative of near-shore conditions off southern California without the use of dispersing agents. Each trophic level appeared to be in a steady-state at the time of first sampling, 5 days after inoculation. Apparent partition coefficients were calculated for each trophic level. The CHC contamination in the diet of the rotifers and anchovy larvae was also calculated. Unfed anchovy larvae accumulated the same amount of CHC as fed larvae and the final concentration appeared to be dependent on the CHC concentration in the seawater. The data in this report suggest that CHC accumulation is not a food-chain phenomenon but rather the result of direct partitioning of the compounds between the seawater and the test organisms.     

Leaf surface compound fromBrassica oleracea (Cruciferae) induces oviposition byPieris brassicae (Lepidoptera: Pieridae)

Summary Chemicals present on the surface of cabbage (Brassica oleracea L.) leaves were extracted by dipping these leaves for 3 s in dichloromethane followed by a 3 s dip in methanol. When offered in dual choice bioassays using green paper cards as a substrate, the methanol extract stimulated oviposition activity byPieris brassicae L. (Lepidoptera: Pieridae) females. The oviposition stimulant was isolated using medium pressure liquid chromatography, reversed-phase HPLC, ion-pair HPLC and ion exchange chromatography. Using¹H-NMR spectroscopy, the stimulant could be identified as glucobrassicin (3-indolyl-methyl-glucosinolate). When pure glucobrassicin was offered at a dose identical to that in the crude methanol extract, butterflies did not discriminate between these two substrates in a dual choice test. It is argued that a high sensitivity for indole glucosinolates as host recognition factors may confer an adaptive value for these specialist crucifer feeders. The nutritional significance of their precursor tryptophan and the non-volatile nature of the aglycones formed upon enzymic hydrolysis in damaged tissues are proposed as properties of indole glucosinolates that contribute to this possible adaptive advantage.     

Evidence that the rotifer<Emphasis Type="Italic"> Brachionus plicatilis</Emphasis> is not an osmoconformer

The rotifer Brachionus plicatilis is euryhaline (growing between 2 and 97 ppt) and has previously been considered an osmoconformer. We suggest that B. plicatilis is an osmoregulator, exhibiting a pattern of Na⁺/K⁺ ATPase activity in response to salinity consistent with that of other osmoregulating euryhaline invertebrates. To examine salinity tolerance, growth rates between 5 and 60 ppt were determined. The activity of Na⁺/K⁺ ATPase was examined, over the same range of salinities, by measuring ATPase activity in rotifer homogenates in the presence and absence of a Na⁺/K⁺ ATPase inhibitor. Maximum specific growth rate (0.95 day^–1) occurred at 16 ppt, highest mean amictic eggs per female (1.41) occurred at 20 ppt, and both parameters decreased rapidly as salinity increased. Egg development time was constant with salinity at 0.92 days. The activity of Na⁺/K⁺ ATPase per milligram protein increased from 3.9 µmol h^–1 at 5 ppt to 6.8 µmol h^–1 at 50 ppt and accounted for 15 and 30% of total ATPase activity, respectively. We suggest that these observations are consistent with increasing stress at high salinities and the occurrence of a hypo-osmoregulatory response. Given the high ATP consumption of Na⁺/K⁺ ATPase at high salinities, it is possible that a proportion of the corresponding decreases in growth rate and egg production are a direct cost of regulation.Communicated by J.P. Thorpe, Port Erin     

Effects of salinity on the clam <Emphasis Type="Italic">Chamelea gallina</Emphasis> haemocytes. Part II: Superoxide dismutase response

The effects of different salinity levels (28, 34 and 40‰) on functional responses of Chamelea gallina haemocytes were evaluated in a two part study dealing with modulations of immune parameters. This part (Part II) of the study was focused on the superoxide dismutase (SODs) activity and expression in haemocyte lysate and cell-free haemolymph. Results of this study established that the exposure of C. gallina specimens at 40‰ salinity provoked a decrease in Mn-SOD and Cu/Zn-SOD activities in haemocyte lysate suggesting a declining superoxide anion generation at the highest salinity tested. Expression of MnSOD was coherent with activity values, while Cu/ZnSOD showed two immunoreactive bands. The former corresponds to the cytosolic Cu/Zn-SOD (16 kDa) was not coherent with the enzyme activity and the second (28–30 kDa) probably attributed to EC-SOD. In cell-free haemolymph, Mn-SOD activity decrease and Cu/Zn-SOD activity increase at 40‰ were observed, likely due to EC-SOD contribution, strongly induced at the same salinity. After EC-SOD detection with two different antibody, we postulate that the EC-SOD like-protein band (29 kDa) may be constitute partly by EC-SOD and probably by Cu/Zn-SOD dimeric form not completely dissociated under reducing condition or a Cu/Zn-SOD degenerated but still recognized by antibody. The cell-free haemolymph increase of EC-SOD at high salinity values plays an important role in immune defence of C. gallina. According to the conclusion of Part I of this study, our data confirmed the destabilizing effect of 40‰ salinity on haemocyte functionality, while to 28‰ exposure, data don’t confirm its stressful action as instead stated by results of Part I. Further studies are necessary to clear up this discrepancy.     

唐鱼卵黄蛋白原的诱导、纯化与鉴定

为探讨利用雄性唐鱼(Tanichthys albonubes)卵黄蛋白原(Vitellogenin,Vtg)作为生物标志物检测环境雌激素的可行性,采用浸浴法用50μg·L-117β-雌二醇(E2)对雄性唐鱼进行染毒,30d后将鱼体整体匀浆进行常规聚丙烯酰胺凝胶电泳(Native-PAGE)以分析Vtg的产生;并采用Mg2+-EDTA选择性沉淀和Q Sepharose阴离子交换层析对Vtg进行分离纯化.结果表明,E2诱导下,雄性唐鱼产生了雌性特异蛋白Vtg,并且可在体内积累;利用Mg2+-EDTA选择性沉淀和Q Sepharose阴离子交换层析的两步纯化方法可分离纯化唐鱼整体匀浆Vtg;经Native-PAGE鉴定,确定唐鱼Vtg的分子量为440kDa.以上结果提示,雄性唐鱼Vtg可以作为环境雌激素监测的有效生物学标记物。     

Some properties of trypsin-like proteases extracted from the seaweedCodium fragile and their purification

Two enzymes were purified from the green algaCodium fragile, collected in 1988 at Fukuoka, Japan, by batch-wise ion-exchange extraction, affinity chromatography and ion-exchange high-performance liquid chromatography (HPLC) using Boc-Ala-Ala-Pro-Arg-pNA as a substrate. The molecular weights of the enzymes were estimated as 38 000 and 39 000, using gel filtration HPLC. The enzymes had the same optimal pH range of 7 to 9 for both activities, and an exclusively hydrolyzed peptide bond on the carboxyl-terminal side of theL-arginine of peptidep-nitroanilides. The ratios of the enzymatic activity for X-Arg-pNA to X-Lys-pNA were larger than 100. The enzymes exhibited 30 times higher activity toward Boc-Ala-Ala-Pro-Arg-pNA when compared with trypsin. The activities were strongly inhibited by diisopropyl phosphofluoridate (DFP), and partially inhibited by phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin and antipain. The isolated enzymes were presumed to be trypsin-like serine protease from their primary substrate specificities and inactivation behavior.     

紫羊茅根中铜结合肽的分离和纯化

利用１次ＳｅｐｈａｄｅｘＧ５０凝胶过滤和２次ＱＡＥＳｅｐｈａｄｅｘＡ２５离子交换层析的方法,从最后６ｄ用ＣｕＣｌ２处理（ρ＝２０ｍｇ／Ｌ）、２０ｄ龄的紫羊茅（Ｆｅｓｔｕｃａｒｕｂｒａｃｖ．Ｍｅｒｌｉｎ）根中分离纯化了１个铜结合肽（ＣｕＢＰ２ａ）,该肽在ＳｕｐｅｒｄｅｘＰｅｐｔｉｄｅＨＲ１０／３０预装柱的ＦＰＬＣ凝胶过滤中在λ２５４ｎｍ处只出现一个峰,经分子量标准曲线估计,其Ｍｒ约为１４００,在ＤｅｌｔａＰａｋＣ１８的反相ＨＰＬＣ中,在λ２５４ｎｍ处也只有１个紫外吸收峰值,这都说明该肽已经达到了较高的的纯度．通过光谱分析,发现该肽的最大紫外吸收值在２４８．１ｎｍ处．对Ｃｕ结合物在植物体内的存在状态进行了讨论．     

Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids

The lipid profiles of a few species of marine unicellular algae and yeast were studied with emphasis on fatty acids as part of a search for the nutritional value of plankton to the diet of marine fish larvae commonly used in marine hatcheries. The general proximate chemistry of rotifers was closely related to the proximate chemistry of the diet organism, exhibiting a higher content of protein and carbohydrate and a lower content of lipid. Major lipids in all algae, yeast and rotifers comprised mono-, di- and tri-glycerides and polar lipids. The algae Chaetoceros gracilis Schutt, Isochrysis galbana Parke and their respective algaefed rotifers exhibited higher amounts of neutral lipids, consisting mainly of cyclic and branched polyunsaturated components. Fatty acid composition of the algae was species-specific, with the highest ratio of polyethylenic to saturated and monoethylenic acid in I. galbana and Phaeodactylum tricornutum Bohlin, and the highest content (15%) of n-3 highly unsaturated fatty acids in Nannochloropsis salina and P. tricornutum. A closely mirrored distribution of the fatty acids, but with a lower amount of n-3 highly unsaturated fatty acids, was present in the respective algae-fed rotifers. Comparison of the fatty acid spectrum of Artemia sp. and Euterpina acutifrons grown in the laboratory on I. galbana with zooplankton samples of E. acutifrons and Oitona nana collected from the sea showed a higher concentration of docosahexaenoic acid (22:6 n-3) in the naturally collected sample. The results indicate that the efficacy of the food algae C. gracilis and I. galbana in increasing the survival of fish larvae in marine hatcheries is not obvious on the sole basis of fatty acid composition.     

双酚A对紫背浮萍生长和光合色素及抗氧化系统影响

以紫背浮萍为实验对象,探究双酚A(bsiphenol A, BPA)对其生长、光合色素含量、抗氧化系统和渗透压调节物质的影响。结果表明,不同浓度(0、1、5、20和50 mg·L~(-1))BPA连续暴露7 d后,与对照组相比,随暴露浓度升高,紫背浮萍湿重和叶绿素含量呈现先显著增加后降低的趋势;暴露组过氧化氢(H_2O_2)含量和抗超氧阴离子自由基活性显著降低;丙二醛(MDA)含量在20和50 mg·L~(-1)组显著升高;暴露组抗氧化酶活性包括超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)和总抗氧化能力(T-AOC)均低于对照组;谷胱甘肽(GSH)含量在高浓度组显著升高,20 mg·L~(-1)组可溶性糖含量显著升高,可溶性蛋白含量在中高浓度组显著增加。研究表明,中低浓度BPA会提高紫背浮萍光合色素含量,有机物积累增多,促进其生长;但随BPA浓度不断升高,紫背浮萍体内产生氧化损伤,抗氧化酶活性及叶绿素含量降低,生长受到抑制。本研究结果将为BPA污染控制及对水生植物生态毒性效应评价提供理论基础。