Evaluation of the copepod Tigriopus californicus as a bioassay organism for the detection of chemical feeding deterrents produced by marine phytoplankton

Marine phytoplankton have been shown to use chemical feeding deterrents to reduce or inhibit zooplankton grazing. In order to screen phytoplankton species for feeding deterrent production and to isolate and identify feeding deterrent compounds, a new, rapid, and reliable laboratory bioassay was developed. This bioassay used the laboratory-reared harpacticoid copepod Tigriopus californicus and measured inhibition of feeding by measuring the fecal pellet production rate. The bioassay was capable of detecting deterrent compounds: (1) adsorbed onto ground fish food (a normally palatable food); (2) dissolved in a mixture of seawater and live Thalassiosira pseudonana cells (a species of diatom which had no feeding deterrent activity); and (3) present in live cell cultures. Method (2) was recommended for use in bioassay-guided fractionation (isolation of chemical compounds), as it was reliable, rapid, accurate, and easy to perform with large numbers of samples. The total bioassay time was < 48 h, and data collection required only a microscope. Methanolic cell extracts of several phytoplankton species were screened for feeding deterrent activity. Extracts from the diatom Phaeodactylum tricornutum and the dinoflagellate Gonyaulax grindleyi gave feeding deterrent responses, while extracts from the diatom Thalassiosira pseudonana gave no feeding deterrent responses. Live P. tricornutum cells deterred feeding at densities of 6x10⁵ cells ml^-1. This bioassay should provide a valuable tool in screening phytoplankton for feeding deterrent compounds and determining the chemical nature of these compounds.     

Growth and grazing rates of the tintinnid ciliate<Emphasis Type="Italic"> Favella taraikaensis</Emphasis> on the toxic dinoflagellate<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis>

Growth and feeding activities of the tintinnid ciliate Favella taraikaensis fed the toxic dinoflagellate Alexandrium tamarense were examined in laboratory experiments. Both growth and ingestion rates of F. taraikaensis as a function of the A. tamarense concentration were fitted to a rectangular hyperbolic equation. The maximum growth and ingestion rates of F. taraikaensis were 1.0 day^–1 and 2.8 cells ind. h^–1 (carbon specific ingestion rates: 3.5 day^–1), respectively, which are both included in the range of previous data reported for Favella spp. feeding on other algae. The gross growth efficiency (GGE) of F. taraikaensis ranged from 0.26 to 0.49 (mean value 0.40) at the concentration of 10–800 cells ml^–1, which is within the range of previous data on Favella spp. Also, the growth and ingestion rates and GGE of F. taraikaensis on A. tamarense were not significantly different from the values on another non-toxic dinoflagellate (Heterocapsa triquetra) at two different prey concentrations. This indicates that the toxicity of A. tamarense probably did not influence the feeding and growth activities of F. taraikaensis at concentrations of less than ca. 800 cells ml^–1. To evaluate the grazing by F. taraikaensis on A. tamarense blooms in the field, the population dynamics of A. tamarense were simulated based on the growth and ingestion parameters of F. taraikaensis. As a result, the grazing impact by F. taraikaensis was considered to potentially regulate the development of A. tamarense blooms. If the toxicity of A. tamarense does not influence the growth and feeding activities of F. taraikaensis, the occurrence of such grazer plankton are considered to be important for predicting the course of A. tamarense bloom dynamics under natural conditions.Communicated by T. Ikeda, Hakodate     

镉和铅对菲律宾蛤仔脂质过氧化及抗氧化酶活性的影响

为了研究亚致死浓度的重金属对海洋贝类的毒性效应,探讨其可能的作用机理,在实验生态条件下以菲律宾蛤仔(Ru-ditapes philip pinarum)为目标生物,采用半静态毒性实验方法,研究了不同浓度Cd2+(0.0948、0.237和0.474mg·L-1)和Pb2+(0.276、0.690和1.380mg·L-1)对菲律宾蛤仔鳃和消化腺组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性以及丙二醛(MDA)含量的影响。结果显示:(1)鳃和消化腺中的抗氧化酶及MDA的变化呈现出类似的趋势,在胁迫初期,各浓度处理组菲律宾蛤仔鳃和消化腺组织SOD和CAT与对照组相比活性都显著升高(P<0.01),呈现出明显的诱导效应,而MDA含量与对照组相比差异不显著(P>0.05);随着胁迫时间的延长,高浓度Cd2+(0.474mg·L-1)和Pb2+(1.380mg·L-1)处理组中SOD、CAT活性快速下降,与对照组相比差异显著;低浓度处理组中的抗氧化活性虽然也较对照组有所下降,但总体下降幅度不如高浓度组明显,并且所历暴露时间较长。各浓度处理组中MDA含量变化一致,均呈现出先升高后降低的趋势,且含量均高于对照组。(2)通过相关性分析,菲律宾蛤仔消化腺组织中的SOD活性与Cd2+浓度的相关性大于鳃组织,与Pb2+浓度的相关性则小于鳃组织;消化腺组织中的CAT活性与Cd2+、Pb2+浓度呈抛物线型相关,与鳃组织CAT活性相关性不十分显著。这说明消化腺组织中SOD活性对Cd2+的敏感性大于鳃组织,消化腺组织中CAT活性对Cd2+、Pb2+的敏感性大于鳃组织。因此,菲律宾蛤仔消化腺中SOD、CAT对水环境中的重金属反应敏感,且存在一定的剂量-效应关系,消化腺组织中SOD和CAT活性与其他敏感性指标一起可以作为指示早期海洋重金属污染的生物标志物。     

Microdistribution of the marine copepodOithona davisae in the shallow waters of Ariake-kai mud flats,Japan

The microdistribution ofOithona davisae Ferrari and Orsi inhabiting shallow waters of Ariake-kai, western Kyushu, Japan, was investigated in 1980–1981 by means of a pumping method. Water samples (10 l) were repeatedly collected from the surface to the bottom at intervals of 0.5 m. This species was dispersed when the tidal current ran fast (more than 20 cm s^–1), but aggregated in the upper layer during the period of slow current. Their density was highest when the current stopped. Aggregation ofO. davisae presumably induced the continuous flat swarm near the surface. It is to be expected that the shallow waters in Ariake-kai would be favorable as nursery grounds for the fish larvae and juveniles which depend on the presence of such swarms.Contribution no. 51 from the Aitsu Marine Biological Station     

Biochemical correlates of dissolved mercury uptake by the oyster Ostrea edulis

From previous work, the equilibrium concentration factor for dissolved mercury in the digestive gland of Ostrea edulis Linnaeus was found to be three to four times higher than that in the gills. In the present study, an analysis of soluble protein revealed values of 49.3±14.2 mg g wet tissue^-1 for the digestive gland and 0.7 ±0.1 mg g wet tissue^-1 for the gills. Starvation significantly reduces the soluble protein level of the digestive gland to 31.1±6.4 mg g^-1 and that of the gills to below the limit of detection. These results suggest that the difference in concentration factors between the gills and digestive gland may be based on a quantitative difference in macromolecular binding sites. However, the uptake of dissolved mercury over a period of 48 h was considerably greater in the gills, so that although the soluble protein content of the tissue may influence the final concentration factor, it does not appear to affect the rate at which this equilibrium is achieved. A more detailed investigation of the mechanism of dissolved mercury uptake by oyster gills has been carried out using isolated tissues. The process is inhibited by 5mM 2–4 dinitrophenol, by the absence of a readily metabolizable substrate (dextrose) in the uptake medium, and by 30mM K⁺. The effect of K⁺ necessitated further investigation with a specific inhibitor of K⁺ transport. Strophanthin G (ouabain), at a concentration of 0.01 mM, caused a significant increase in mercury uptake.     

Influence of a botanical pesticide,azadirachtin, on ultimobranchial gland of the freshwater catfish Heteropneustes fossilis

Freshwater catfish, Heteropneustes fossilis, were subjected to 42 mg L^?1 of azadirachtin for short-term exposure (96 h) and to 10.5 mg L^?1 for long-term exposure (28 days). Six fishes were sacrificed on each time interval from control and experimental groups after 24, 48, 72, and 96 h in the short-term exposure and after 7, 14, 21, and 28 days in the long-term experiment. The calcium regulating endocrine gland, viz. the ultimobranchial gland, was fixed for histological studies. Up to 72 h of azadirachtin treatment, there was no histological alteration in the UBG (ultimobranchial gland) of H. fossilis. After 96 h exposure, the ultimobranchial cells stain feebly and the nuclear volume of these cells were slightly decreased. No histological alterations have been observed in the UBG cells up to 14 days following azadirachtin exposure. After 21 days, the cytoplasm of ultimobranchial cells was feebly stained and the nuclear volume exhibits a decrease. The nuclear volume of these cells was further decreased and the gland shows vacuolization and degeneration at certain places after 28 days azadirachtin treatment. Hence, it can be concluded that azadirachtin severely affects the physiology of calcium homeostasis in H. fossilis. Therefore, the botanical pesticides should be used carefully near aquatic reservoirs.     

Feeding and swimming of lysianassid amphipods in a shallow cold-water bay

The potential for dispersal by lysianassid amphipods and their localization to carrion in a shallow cold-water bay in the Middle Saint Lawrence Estuary were assessed by means of endobenthic sampling, SCUBA observations, measures of swimming speeds, and by exposure of bait (50–100 g of fish) in traps. Seventy-five to 99.9% of animals attracted to traps were lysianassid amphipods belonging to five species. Lysianassid species were spatially segregated in the Bay at low tide but all were more or less dispersed at high tide. Second cohortAnonyx sarsi Steele and Brunel,Boeckosimus edwardsi andOnisimus littoralis (Krøyer) were more dispersed than the small first cohort individuals. Second cohortA. sarsi were crawlers or low (0–0.5 m off the bottom) suprabenthic swimmers in the day, but upper (0.5–2 m) suprabenthic swimmers at night. In contrast, first cohortA. sarsi were crawlers or low suprabenthic swimmers day-and-night, whileOrchomenella pinguis (Boeck) followed this swimming pattern at night but were generally akinetic in the day. Mean swimming speeds ofA. sarsi (13.6 cm s^-1) andOn. littoralis (12.1 cm s^-1) were 2 to 3 times greater than those ofOr. pinguis (7.4 cm s^-1) andPsammonyx nobilis (Stimpson) (4.4 cm s^-1). Catchability coefficients (i.e. ratio number of individuals per trap:endobenthic abundance) were 74 (A. sarsi), 8 (On. littoralis), 7 (Or. pinguis), and 0.7 (P. nobolis) m² of bottom. Gut content analysis indicated thatA. sarsi fed mostly on large carrion, whileOn. littoralis were markedly opportunistic, andOr. pinguis andP. nobilis relied on detritus, algae, and small crustaceans.     

Toxicity and attraction effects produced byMorinda citrifolia fruits on theDrosophila melanogaster complex of species

Summary The ripe fruit of the Indian mulberry,Morinda citrifolia, is the host plant forDrosophila sechellia but is highly toxic for three closely related species (D. melanogaster, D. simulans, D. mauritiana). A simple bioassay is described with which a clear dose response to the fruit was found for these species. Significant differences in reactivity to the ripe fruit were found among species. Tested strains ofD. simulans andD. mauritiana adults were more sensitive to the toxic properties of the fruit thanD. melanogaster. A marked intraspecific variability was shown inD. melanogaster. Reciprocal interspecific hybridizations betweenD. sechellia andD. mauritiana suggested an autosomal dominant control of resistance. MoreoverD. melanogaster intraspecific crossings suggested the influence of an additional X-linked factor. Responses of flies toMorinda fruit in different states were tested in a T olfactometer. The less resistant strains ofDrosophila generally showed less preference for the ripe fruit.     

Vertical distributions and photosynthetic action spectre of two oceanic picophytopianlcters,Prochlorococcus marinus andSynechococcus sp.

Two picophytoplankters,Prochlorococcus marinus andSynechococcus sp., were isolated from the bottom of the euphotic zone (150 m depth) in the western Pacifie Ocean. The concentration ofP. marinus at this depth was more than 10⁴ cells ml^–1 while that ofSynechococcus sp. was less than 10² cells ml^–1. TheP. marinus isolate has a high divinyl-chlorophylla:b ratio similar to that of the Mediterranean strain, while theSynechococcus sp. isolate is of the phycourobilinrich type. The growth rate ofP. marinus was higher thanSynechococcus sp. when both were cultured under weak blue-green to blue-violet light (ca. 2 E m^–2 s^–1). While the chlorophyll-specific absorption spectra showed higher values inSynechococcus sp., the photosynthetic action spectre revealed thatP. marinus was able to use blue-violet light, whereasSynechococcus sp. was able to use blue-green light, more efficiently for photosynthesis. The photosynthetic quantum yield ofP. marinus was higher than that ofSynechococcus sp. at any wavelength between 400 and 700 nm. The calculated in situ photosynthesis rates per Gell volume forP. marinus were estimated to be higher than forSynechococcus sp. at 50 and 150 m depth. These results indicate thatP. marinus photosynthetically surpassesSynechococcus sp. in the blue-light-rieh environment of the oceanic euphotic zone. This may be why the former predominates at depths in temperate to tropical open ocean waters.     

Effect of electrokinetic transport on the vulnerability of PAH-degrading bacteria in a model aquifer

There has been increasing interest in employing electro-bioremediation, a hybrid technology of bioremediation and electrokinetics, to overcome the low bioavailability of hydrophobic organic contaminants (HOC) by homogenizing sorption-retarded HOC and immobilised microorganisms. Present electro-remediation approaches mainly aim at macroscale pollutant extraction and tend to neglect possible impacts of direct current (DC) on the physiology of microorganisms. The effect of weak electric fields (X = 1 V cm⁻¹) on the fitness of electrokinetically dispersed fluorene-degrading Sphingomonas sp. LB126 in bench-scale model aquifers was investigated by flow cytometry using propidium iodide (PI) as an indicator that distinguishes between PI-permeable (cells with porous membranes, i.e. dead or vulnerable) and PI-impermeable bacteria. After 15.5 h of DC treatment 56% of all cells recovered were dispersed at the centimetre scale relative to 29% in the absence of DC. There was no overall negative effect of the 15.5-h DC treatment on cell vulnerability, as 7.0% of the DC-treated bacteria exhibited PI-staining compared to 6.5% of the control population. Minor differences were observed in the subpopulation that had been mobilised by electroosmosis with an approximately twofold increase in the percentage of PI-stained cells relative to the control. Enhanced PI staining did not correlate with reduced culturability of the cells on rich-medium agar plates. Relative to the control, DC-treated cells mobilised by electroosmosis were threefold more culturable, confirming earlier data that that PI-cell membrane permeability does not always indicate reduced viability of oligotrophic environmental bacteria. Our findings suggest that electrokinetics is a valuable mechanism to transport viable and culturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria in soil or sediments.     

Petroleum hydrocarbons in the marine bivalve Venus verrucosa: accumulation and cellular responses

Cellular and subcellular responses of the marine burrowing bivalve Venus verrucosa collected from the north-eastern coastline of Malta from January to June 1985, after exposure to petroleum hydrocarbons (PHC) were investigated. After long-term exposure to 100 gl^-1 of water-accomodated fractions (WAF) of crude oil, PHC were found to accumulate most rapidly in the digestive gland and then in the gills, with saturation levels being reached within 100 d of exposure in both cases. PHC accumulation, both in the mantle and muscle tissues, was more gradual and consistent throughout the whole exposure period. After 150 d of exposure, the digestive cells of the digestive gland were significantly reduced in height (atrophy) and exhibited reduced lysosomal membrane stability. After 144 h of exposure to higher concentration of PHC (820 and 420 gl^-1), several cytological effects were recorded, including an increase in cell volume and activity of gill mucocytes as well as in the number of haemocytes in gill blood sinuses. There was also evidence of damage to the epithelial lining of the foot, stomach and style sac and marked atrophy of the digestive cells of the digestive gland. The significance of such responses is discussed.     

Ontogenetic changes in digestive enzyme activity of the spiny lobster,<Emphasis Type="Italic"> Jasus edwardsii</Emphasis> (Decapoda; Palinuridae)

Digestive enzyme profiles of puerulus, post-puerulus, juvenile and adult stages of the spiny lobster Jasus edwardsii Hutton were determined in order to identify ontogenetic changes in digestive capabilities and assess capacity to use dietary components and to exploit diets to meet nutritional requirements. Juvenile and adult lobsters exhibited a diverse range of enzymes, suggesting they can exploit varied diets. Proteolytic activity of trypsin, chymotrypsin and carboxypeptidase A (1.86–3.70 U mg^–1 digestive gland protein) was significantly higher than carbohydrase activity of amylase, -glucosidase, cellulase and chitinase (0.0014–0.38 U mg^–1 digestive gland protein), thus showing that dietary protein was more important than carbohydrate and that the lobster is carnivorous. These conclusions are consistent with other studies that found spiny lobsters to feed predominantly on crabs, bivalves, ophiuroids and sponges. Lipase activity (0.371 U mg^–1 digestive gland protein) was also relatively high, thus showing the importance of dietary lipid. Total activities (units per digestive gland) of every enzyme assayed increased significantly with lobster carapace length. There were few significant ontogenetic trends in specific enzyme activity (U mg^–1 digestive gland protein). Amylase and laminarinase specific activity was significantly higher in small lobsters than large lobsters (regression analyses, respectively, F_(1,23)=9.84, P=0.005; F_(1,11)=19.65, P=0.001), suggesting that carbohydrates including laminarin are more important in the diet of small juveniles. Trypsin, amylase and lipase activities were detected in all non-feeding puerulus stages, suggesting that feeding is not a cue for digestive enzyme production in J. edwardsii. Significant variations in total and specific activities of amylase (F_(1,3)=14.2, P=0.00; F_(1,3)=14.2, P=0.00) and trypsin (F_(1,3)=8.8, P=0.00; F_(1,3)=21.41, P=0.00) and a declining trend in lipase total activity between non-feeding puerulus stages suggests that they may be hydrolysing endogenous energy reserves to sustain their onshore swimming activity prior to settlement. Profiles suggest carbohydrate and lipid are utilised first, followed by protein. Consistently high levels of lipase in all puerulus stages (0.24–0.7 U digestive gland^–1; P>0.05) confirm the importance of lipid as a major energy substrate.Communicated by G.F. Humphrey, Sydney     

Localisation and surface quantification of secondary metabolites in the red alga Delisea pulchra

The localisation of halogenated furanones, the biologically active secondary metabolites from the red alga Delisea pulchra (Greville), was determined by a combination of fluorescence microscopy, culture studies and quantitative chemical analyses. All types of evidence showed that furanones are localised in the central vesicle of gland cells in D. pulchra. These cells release furanones onto the surface of the plant, where they can be quantified using a newly developed surface extraction-technique. Levels of furanones on the surface of the plant were highest near the apical tips (≃100 ng cm⁻²), and decreased towards the base of the alga. Variation in furanone levels within the plant and variation in the number of gland cells followed a similar pattern. The localisation of furanones within gland cells in D. pulchra and the presence and concentrations of furanones on the surface of the plant are consistent with furanones functioning as antifoulants and in mediating other ecological interactions at the surface of the alga. Received: 5 March 1998 / Accepted: 10 September 1998     

全氟辛基磺酸及其替代产品的内分泌干扰效应评价

为研究全氟辛基磺酸(PFOS)及其替代品的内分泌干扰效应,将重组人雌激素/雄激素/孕激素/甲状腺激素/维甲酸受体基因的双杂交酵母暴露于不同浓度的PFOS及替代品中,并结合体外代谢活化技术检测直接和间接的类/抗激素活性。结果表明,被替代品PFOS表现为雄激素受体、甲状腺受体和维甲酸受体拮抗剂,且在代谢活化后新增了雌激素受体拮抗效应,其半数效应浓度(EC₅₀)在3.0×10^-4～4.8×10^-3 g·L^-1之间;同时,4种替代化学品没有任何效应检出,说明不会通过与核受体结合干扰天然激素的正常功能。研究结果说明,与原化学品相比,替代产品的应用在内分泌干扰效应方面可能具有更低的环境风险。     

Carotenoids in the tiger prawn Penaeus esculentus during ovarian maturation

Female Penaeus esculentus Haswell were collected by 15 to 20 min duration trawls during 1990. Carotenoids were analysed in the digestive gland, abdominal muscle, the remainder of the body (hereafter called integument) and ovary of prawns in Stage 2 through Stage 4 (fully mature) of maturation. The only oxycarotenoids (xanthophylls) identified were astaxanthins or astaxanthin esters; occasionally low levels of -carotene were detected in the digestive gland. The concentrations of astaxanthin monoesters (AM) and diesters (AD) were highest, with only minor amounts of free astaxanthins (Ast), except in the maturing ovaries, where free astaxanthins predominated (up to 80% of the total carotenoid). Of the total carotenoid, 82 to 94% was in the integument, but at maturity the digestive gland contained 10.7±3.4% and the ovary 5.6±0.9% of the total carotenoid. Only the ovary increased in mass during maturation, reaching up to 5.2% of total prawn mass. During this period, digestive gland concentrations of AM, AD and Ast all increased (tota 20 to 120 g g^-1); levels in the muscle and integument varied little throughout maturation (total 0.4 and 100 g g^-1, respectively); ovary AM levels remained low throughout (1.5 to 1.2 g g^-1), AD increased from only 2 to 5 g g^-1, but Ast increased from 2 to 34 g g^-1. Apart from the ovary, AM concentrations were the most variable. In common with other decapod Crustacea, the maturing ovary of P. esculentus contained high levels of carotenoids, indicating that these may have an important role in early development. The natural diet of P. esculentus includes a variety of carotenoids, but except for a little -carotene, the digestive gland, where absorption occurs, contained astaxanthins, with only an occasional trace of -carotene. This suggests that the conversion of dietary carotenoids to astaxanthin occurs soon after ingestion.     

Acid and alkaline phosphatase activities in the clam Ruditapes philippinarum

Acid and alkaline phosphatase activities have been partially characterized in Ruditapes philippinarum (Adams and Reeve, 1850). Two activity peaks at pH=4.5 and pH 10.5 were detected in the gill, digestive gland, mantle, siphon and foot. Acid phosphatase activity was higher than that of alkaline phosphatase. The highest activity for both enzymes was observed in the digestive gland and, in decreasing order, the gill, foot, siphon and mantle. Alkaline phosphatase activity was similar in the mantle, siphon and foot. K _m values were determined for both enzymes in the gill and digestive gland. Hill coefficients were near 1, indicating no allosteric behaviour for either enzyme in the two organs. The optimum temperature was the same for acid phosphatase in both gill and digestive gland (50 °C), while for alkaline phosphatase it differed for these two organs (gill, 40 °C; digestive gland, 35 °C). The apparent activation energy was obtained from Arrhenius plots, and ranged from 8.61 kcal/mol for alkaline phosphatase in the gill, to 10.84 kcal/mol for acid phosphatase in the digestive gland. The effects of metals (1 mM conc) on both enzyme activities were assayed in vitro. Hg strongly inhibited the enzyme activities in the gill and digestive gland, probably because of its affinity for the sulphydryl group. Histochemically, acid phosphatase in the gill was located in a granular form throughout the gill cells, but was undetectable in the ciliate epithelium of the gill filaments. Alkaline phosphatase was located in the gill skeleton. Clam size and phosphatase activities were inversely related, probably reflecting a decrease in shell deposition with inereasing size. As a function of season, both enzymes were present in lowest amounts in winter, when undifferentiated sex cells were predominant in the germinative epithelium, and highest in summer, when ripe individuals of both sexes were more frequent.     

Some properties of calcium-activated adenosine triphosphatase from the hermatypic coralGalaxea fascicularis

Samples of the hermatypic coralGalaxea fascicularis were collected between April 1987 and April 1990 from coral reefs off Singapore (103 °45E; 1 °13N). Ca²⁺-activated adenosine triphosphatase (ATPase) activity was detected in the plasma-membrane-enriched heavy microsomal fraction ofG. fascicularis. The high affinity component hadKm andVmax values of 0.0021 mM and 0.050 µmol Pi mg^–1 protein min^–1, respectively; corresponding values for the low affinity component were 0.15 mM and 0.85 µmol mg^–1 protein min^–1. The activity of the high affinity component was inhibited 80 and 50%, respectively, by the anticalmodulin drugs calmidazolium and chlorpromazine. The low affinity component of the Ca²⁺-ATPase may represent activities of alkaline phosphatase, Ca²⁺-ATPase from membranes of mitochondria and endoplasmic reticulum, or calmodulin-dissociated plasma membrane Ca²⁺-ATPase resulting from the removal of Ca²⁺ by EDTA during the isolation process. The high affinity Ca²⁺-ATPase is probably the enzyme responsible for Ca²⁺ extrusion from the cells ofG. fascicularis. The high and low affinity components of this Ca²⁺-ATPase could use ATP and ADP as substrates. Maximum activities of both components were registered at pH 7 and at 45°C. Ruthenium red, a specific inhibitor of Ca²⁺-ATPase, inhibited the activities of the high and low affinity Ca²⁺-ATPase by 100 and 60%, respectively. Inhibition of the activities of both components was also observed with sulphydryl reagents (PCMB and mersalyl). However, DCMU, diamox, dinitrophenol, iodoacetate, fluoride, cyanide, ouabain, oligomycin B and L-phenylalanine had no effect on the enzyme activities.     

Nutrient starvation effects on the allelochemical potency of Alexandrium tamarense (Dinophyceae)

Batch culture experiments were performed to investigate potential effects of nutrient starvation on the allelochemical potency of the toxic dinoflagellate Alexandrium tamarense. Triplicate cultures with reduced nitrate (−N) or phosphate (−P) seed were compared to nutrient-replete (+N+P) cultures. Total depletion of the dissolved inorganic limiting nutrient, reduced cell quotas, changed mass ratios of C/N/P and reduced cell yield clearly indicate that treatment cultures at stationary phase were starved by either N or P, whereas growth cessation of +N+P cultures was probably due to carbon limitation and/or a direct effect of high pH. Pulsed addition of the limiting nutrient allowed −N and −P cultures to resume growth. Lytic activity of A. tamarense as quantified by a Rhodomonas bioassay was generally high (EC₅₀ around 100 cells mL⁻¹) and was only slightly modulated by growth phase and/or nutrient starvation. Lytic activity per cell increased with time in both +N+P and −P cultures but not −N cultures. P-starved stationary-phase cells were slightly more lytic than +N+P cultures, but this difference may be due to increased cell size and/or accumulation of extracellular compounds. In conclusion, only slight changes but no general and major increase in lytic activity in response to nutrient starvation was observed.     

Quantitative and ultrastructural studies on the reproductive biology of the polychaete Pholoe minuta in Galway Bay

Previous works by the present authors have identified, in a qualitative fashion, the reproductive cycle of Pholoe minuta (Fabricius) in inner Galway (53°13.5N; 9°7W). This quantitative, histological study, carried out over the period late May 1981 to late April 1982, shows that P. minuta is polytelic (two to three annual spawnings in an individual's lifespan). Individuals become sexually mature for the first time when approaching 3 yr of age. Estimates were made of the size of the reproductively active population (47 to 49%), sex ratio (1:1), duration (10 to 11 mo) and pattern (unimodal) of gametogenesis and fecundity levels (210 198 eggs m^-2). Maturation (mean oocyte size=85 m) and spawning (for 1 wk during late March through early April) were shown to be synchronised. Throughout oogenesis, follicle cells play an important role in binding together oocyte clusters, and desmosomal contacts are evident between oocyte and follicle cell plasmalemmas. No evidence for the synthesis of yolk precursors in the follicle cell cytoplasm was discovered. Oocytes are rich in Golgi apparatuses (yolk synthesising) and nuclear pores are numerous. Rough endoplasmic reticulum is distended in nature. Egg envelope development does not appear to commence until after the onset of vitellogenesis. Mature spermatozoa are of the primitive bullet-shaped type, with an unmodified acrosome. A possible method of elevation membrane formation is described.     

Calculating carbon biomass ofPhaeocystis sp. from microscopic observations

Conversion factors for calculating carbon biomass ofPhaeocystis sp. colonies and free-living cells were determined from microscopic observations and chemical analysis conducted on cultured and naturalPhaeocystis sp. populations originating from the Southern Bight of the North Sea in 1986 and 1987. They allow calculation, in terms of carbon biomass, of the different forms ofPhaeocystis sp. that succeed each other when the population is growing, on the basis of microscopic observations. The latter include enumerations of free-living cells (flagellated and non-motile) and colonies, as well as colonial biovolume measurement. Specific application to natural populations from Dutch coastal waters during spring 1986 shows that more than 90% ofPhaeocystis sp. carbon biomass is under colonial form, most of it exceeding the grazing characteristics of current zooplankton at this period of the year. Detailed analysis of seasonal changes shows in addition that the size of the colonies greatly increases during the course ofPhaeocystis sp. flowering, reaching sizes as high as 1 mm diameter at the top of the bloom when nutrients are depleted. Physiologically this corresponds to an enhanced synthesis of mucilaginous substances, with the decrease of available nutrients leading to an increasing contribution of the matrix to the total colonial carbon during the course of the bloom. Carbon content ofPhaeocystis sp. colonies therefore greatly varies with their size, ranging from 0.3 to 1430 ngC colony^–1.