Electro-immunoblotting characterization ofPseudo-nitzschia multiseries andP. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producingPseudonitzschia multiseries (=Pseudo-nitzschia pungens f.multiseries) and the non-toxicP. pungens (=P. pungens f.pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared againstP. multiseries strongly labelled the high molecular-weight antigens of allP. multiseries strains tested and showed little reactivity towardsP. pungens extracts on Western blots.P. pungens antibodies strongly labelled high molecular-weightP. pungens antigens and faintly labelled a fewP. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxicP. multiseries and non-toxicP. pungens are high molecular-weight (°>100kDa) polysaccharides located on the surface of these diatoms.Author for correspondence     

Seasonal variation in egg morphology and hatching success in the calanoid copepods Temora longicornis, Acartia clausi and Centropages hamatus

We report here on the seasonal changes in ultramicroscopic morphologyof the eggs of the calanoid copepod Temora longicornis. Duringa field study between 1996 and 1998 in the Menai Strait, easternIrish Sea, T. longicornis produced two types of eggs; hatchingor subitaneous eggs were spawned during autumn–winter,whereas a high proportion of non-hatching eggs (up to     

Formation of domoic acid and fatty acids inPseudonitzschia pungens f.multiseries with scale of culture

Pseudonitzschia pungens f.multiseries was cultured in 20-L polycarbonate carboys, 350-L fibreglass columns and 500-L plastic bags to determine the effects of medium enrichment and scale of culture on cell yield, production of cellular domoic acid and formation of fatty acids, particularly the potential tracer acid 16:4n-1. Cell concentrations were highest in seawater enriched with stock levels of nitrate and phosphate, but with double the stock level of silicate, at all scales of culture. Cellular toxin in 20, 350 and 500-L cultures averaged 0.32, 0.04 and 2.56 pg cell^-1 and was independent of medium used. The order of magnitude difference in levels of cellular toxin was considered to reflect the varying levels of irradiance within the culture vessels. Support was given to this by the significant difference in content of total cellular fatty acids, due principally to the algal storage acid 16: 1n-7, which is known to be influenced by irradiance. Levels of cellular domoic acid correlated significantly with total fatty acids in 350 and 500-L cultures. Bag cultures producing significantly higher levels of cellular domoic acid provided lower relative proportions of 16:4n-1, which limited its use as a tracer for food-web studies.     

Feeding behaviour and migrations in a natural population of the copepod Acartia tonsa

This paper deals with the variations on feeding activities and diel migrations of Acartia tonsa Dana, the dominant copepod species in Berre lagoon (west Mediterranean French coasts).A 27 hour in situ study was carried out during June 1989, at a station located in the south west of the lagoon. Vertical profiles of salinity, temperature and dissolved oxygen taken each 12 hours showed a stratification of the water column in two distinct layers: (1) a superficial layer with higher temperature, moderate salinity, and high oxygen concentrations; (2) a colder, more saline and almost anoxic deep layer. In situ chlorophyll a measurements were made at –1, –3 and –5 m; concentrations were relatively homogeneous through the water column during the whole sampling period.Zooplankton samples were collected every 3 hours with a 200 µm mesh net, in three strata (0–2 m, 2–4 m, 4–6 m). A complete dominance of A. tonsa was observed in the zooplankton community. Our results point out clearly a nocturnal migration, with individuals concentrating in both superficial layers; thus an unimodal migratory pattern can be inferred. Gut flourescence measured following the Mackas & Bohrer technique (1976), showed higher values during night time, and values for females were the highest with wider day—night variations. Similar results were found in laboratory experiments with copepods fed with a culture of Dunaliella tertiolecta.Gut evacuation rate was measured in two laboratory experiments either mixing or separating males and females. Evacuation rate was 17.92 and 27.25 min for males and females respectively.Phytoplankton daily ration for A. tonsa calculated by gut flourescence and gut evacuation rate was particularly low, for it represents only 10% of the individual carbon weight.Moreover, grazing impact on phytoplankton is very restricted, it represents less than 1% of the daily phytoplankton stock.     

Host-specific adaptation governs the interaction of the marine diatom,Pseudo-nitzschia and their microbiota

The association of phytoplankton with bacteria is ubiquitous in nature and the bacteria that associate with different phytoplankton species are very diverse. The influence of these bacteria in the physiology and ecology of the host and the evolutionary forces that shape the relationship are still not understood. In this study, we used the Pseudo-nitzschia–microbiota association to determine (1) if algal species with distinct domoic acid (DA) production are selection factors that structures the bacterial community, (2) if host-specificity and co-adaptation govern the association, (3) the functional roles of isolated member of microbiota on diatom–hosts fitness and (4) the influence of microbiota in changing the phenotype of the diatom hosts with regards to toxin production. Analysis of the pyrosequencing-derived 16S rDNA data suggests that the three tested species of Pseudo-nitzschia, which vary in toxin production, have phylogenetically distinct bacterial communities, and toxic Pseudo-nitzschia have lower microbial diversity than non-toxic Pseudo-nitzschia. Transplant experiments showed that isolated members of the microbiota are mutualistic to their native hosts but some are commensal or parasitic to foreign hosts, hinting at co-evolution between partners. Moreover, Pseudo-nitzschia host can gain protection from algalytic bacteria by maintaining association with its microbiota. Pseudo-nitzschia also exhibit different phenotypic expression with regards to DA production, and this depends on the bacterial species with which the host associates. Hence, the influences of the microbiota on diatom host physiology should be considered when studying the biology and ecology of marine diatoms.     

Feeding of Acartia tonsa Dana (Copepoda,Calanoida): predation on nauplii of Canuella perplexa T. et A. Scott (Copepoda,Harpacticoida) in the sluice-dock at Ostend

Feeding experiments in which the zooplankton fraction of the Sluice-dock smaller than 200 μm is offered to Acartia tonsa, the dominant calanoid of this biotope, show that it feeds on the nauplii of Canuella perplexa, the dominant benthic harpacticoid. The ecological implications of this carnivorous feeding, occurring in the presence of natural phytoplankton concentrations, are briefly discussed.     

Interaction between bacteria and the domoic-acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle; can bacteria produce domoic acid autonomously?

The interaction between bacteria and phytoplankton is increasingly becoming recognised as an important factor in the physiology of toxin production and the dynamics of harmful algal blooms (HABs). Bacteria can play a direct or indirect role in the production of biotoxins once solely attributed to microalgae. Evidence implicating bacteria as an autonomous source paralytic shellfish poisoning biotoxins raises the question of autonomous bacterial toxigenesis of the neurotoxin domoic acid (DA), the cause of amnesic shellfish poisoning. Here, we examine whether the previously observed bacterial enhancement of DA production by Pseudo-nitzschia multiseries (Hasle) Hasle may be attributable to independent biotoxin production by the extra-cellular bacteria associated with this diatom. The growth and toxicity of six cultures of xenic P. multiseries clone CLN-1 were followed for 24 days. Up to day 14 (mid-stationary phase), DA production was not statistically different among culture flasks. On day 14, P. multiseries cells were removed by gentle filtration from a set of triplicate flasks, leaving the bacteria in the filtrate. Following the removal of the algal cells, DA in the filtrate ceased to increase. Instead, DA levels continuously declined. A follow-up experiment determined that this was likely caused by photodegradation rather than by bacterial degradation. We conclude that after removing P. multiseries cells, the extra-cellular bacteria remaining in the filtrate were incapable of autonomous DA toxigenesis, even in the presence of P. multiseries exudates. However, scanning electron microscopy revealed that P. multiseries cells harboured epiphytic bacteria, the importance of which can still not be ruled out in DA production.     

Acclimation of the Marine Diatom Pseudo-nitzschia australis to Different Salinity Conditions: Effects on Growth,Photosynthetic Activity,and Domoic Acid Content1

Toxic Pseudo-nitzschia australis strains isolated from French coastal waters were studied to investigate their capacity to adapt to different salinities. Their acclimation to different salinity conditions (10, 20, 30, 35, and 40) was studied on growth, photosynthetic capacity, cell biovolume, and domoic acid (DA) content. The strains showed an ability to acclimate to a salinity range from 20 to 40, with optimal growth rates between salinities 30 and 40. The highest cell biovolume was observed at the lowest salinity 20 and was associated with the lowest growth rate. Salinity did not affect the photosynthetic activity; F_v/F_m values and the pigment contents remained high with no significant difference among salinities. An enhanced production of zeaxanthin was, however, observed in the late stationary and decline phases in all cultures except for those acclimated to salinity 20. In terms of cellular toxin content, DA concentrations were 2 to 3-fold higher at the lowest salinity (20) than at the other salinities and were combined with a low amount of dissolved DA. The fact that P. australis accumulate more DA per cell in less saline waters, illustrates that climate-related changes in salinity may affect Pseudo-nitzschia physiology through direct effects on growth, physiology, and toxin content.     

Effect of UVB radiation on the survival, feeding, and egg production of the brackish-water copepod, Sinocalanus tenellus, with notes on photoreactivation

We examined the effects of UVB radiation on hatching success of eggs, survival of various naupliar and copepodite stages, and feeding and egg production of adult females of the brackish-water copepod, Sinocalanus tenellus, by exposure to varying doses of UVB irradiance in the laboratory. Artificial UVB radiation resulted in an increased mortality of eggs, nauplii and copepodites with increasing UVB doses. UVB induced damage was stage-specific with eggs being most susceptible (LD₅₀= 4.1 kJ m^–2 ) and adult females being least susceptible (LD₅₀= 16.7 kJ m^–2). Effects on feeding and egg production of adult females were significantly evident at UVB doses higher than 11.0 kJ m^–2 and 7.0 kJ m^–2, respectively. We also examined the photorepair response of eggs and various developmental stages in simultaneous irradiation of UVB and enhanced PAR. With enhanced PAR there was a considerable recovery against UVB damage, being higher for younger animals than older ones. In nature, however, solar UVB radiation may rarely cause appreciable damage to S. tenellus population due to optically high attenuation properties of their habitat waters.     

Diversity and distribution of epibiotic bacteria on Pseudo-nitzschia multiseries (Bacillariophyceae) in culture, and comparison with those on diatoms in native seawater

Previous studies on the interaction between bacteria and harmful algal bloom species have mostly considered the bacteria in the bulk solution. Here, we document the abundance and mode of attachment of bacteria growing on the cell surface of the domoic acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle in culture, compared with diatoms in field samples. The epiphytic bacteria were examined by scanning electron microscopy to visualize their morphology and mode of attachment. Two P. multiseries cultures were studied: clone CLN-1 and sub-clone CLN-1-NRC; the latter had been maintained in another laboratory for 2 years. Each of these P. multiseries cultures exhibited a clearly different assemblage of epibiotic bacteria, even though both originated from the same parent culture. The bacterial diversity was greater in clone CLN-1 (nine distinct morphotypes seen) than in sub-clone CLN-1-NRC (six morphotypes). The former clone also produced more domoic acid than the latter. There was a succession of bacterial morphotypes as well as an increase in the number of epiphytic bacteria per diatom cell during the progression from exponential to stationary phase. The most diverse and common morphotypes were rod-shaped cells (e.g. a Caulobacter-like bacterium attached by a discoid holdfast). Epibionts showed a preference for attachment at specific regions of the host diatom frustule, e.g. the raphe or cingulum, locations where organic matter may be extruding from the diatom cell. Most diatom cells carried only one to five bacteria, and up to ca. 60% of the intact diatom cells (although intact cells themselves were infrequent) were still free of epibiotic bacteria at the end of the 31-day batch culture experiment. Sequencing of the SSU rRNA gene showed that five of the eight bacterial strains isolated from the P. multiseries cultures were members of the Alphaproteobacteria, three of the Gammaproteobacteria and one of the Bacteroidetes. A morphologically diverse assemblage of epibiotic bacteria was also found on both centric and pennate planktonic diatoms in natural coastal waters. Of the eight morphotypes recorded, all but two were also found in the cultures. Relatively fewer wild diatom cells carried bacteria compared to cells in culture. We hypothesize that the diversity and abundance of epiphytic bacteria may explain some of the variability seen in the production of DA by different P. multiseries clones, and should be considered as another important and controllable variable that influences diatom cell physiology.     

Release and degradation of amnesic shellfish poison from decaying Pseudo-nitzschia multiseries in presence of bacteria and organic matter

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at least 20 days with 1× or 4× bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a potential future mitigation technique.     

Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: morphology,toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species

The genus Pseudo-nitzschia includes several species capable of producing domoic acid, the causative agent of Amnesic Shellfish Poisoning. Some of these species have been recorded frequently in the Gulf of Naples. For one of the species, P. multistriata, which has been recurrently found in our sampling area since 1995, this is the first report for European waters. Here we provide further details on the fine structure of this species. Pseudo-nitzschia multistriata was the only one found to produce domoic acid among all the Pseudo-nitzschia species from the Gulf of Naples, and this finding raises the number of potentially toxic species in this genus to nine. Phylogenetic relationships among several Pseudo-nitzschia species were assessed using the hypervariable domains (D1–D3) of the large subunit (LSU) rDNA. The match between the phylogeny obtained and important taxonomic characters used in this genus are discussed. Results show that P. multistriata clusters with wider species lacking a central larger interspace in the raphe. Close genetic relationships were determined between P. fraudulenta and P. subfraudulenta, and between P. pungens and P. multiseries. Genetic differences among these pairs of species are comparable to those among isolates of P. pseudodelicatissima from the Gulf of Naples, indicating high intraspecific genetic diversity of Pseudo-nitzschia species in the relatively conserved LSU region. This could explain the problematic results obtained when testing a match between species-specific Pseudo-nitzschia LSU probes and our sequences.     

Comparative carbon-specific ingestion rates of phytoplankton by Acartia tonsa,Centropages velificatus and Eucalanus pileatus grazing on natural phytoplankton assemblages in the plume of the Mississippi River (northern Gulf of Mexico continental shelf)

During four cruises in continental shelf waters of the northern Gulf of Mexico in the winters of 1981–83, we performed quantitative studies on the grazing of the copepods Acartia tonsa, Centropages velificatus, and Eucalanus pileatus, on phytoplankton using natural particulate assemblages as food. Stations were in, or adjacent to the plume of the Mississippi River, thereby providing wide spectra of phytoplankton and suspended riverine particulate concentrations. Phytoplankton cell volume was converted to carbon, and this, coupled with carbon content measurements of these three copepod species, allowed comparisons of daily ingestion effort even though the copepods were of different sizes. Data were expressed in the same units (% of copepod body carbon ingested copepod ^–1 d^–1) for each species. Over similar ranges of phytoplankton carbon concentrations (0.21–92.06 gCl^–1), Acartia tonsa had higher carbon-specific ingestion rates (x = 22.31%, range = 0.08–152.37%) than C. velificatus (x = 2.8%, range = 0.00–31.09 %) or E. pileatus (x = 1.27%, range = 0.10–2.80%). Carbon-specific ingestion rates increased with increasing phytoplankton carbon concentration for A. tonsa (R² = 0.81) and there was no evidence of saturated feeding on the carbon concentrations offered. A similar, but weaker trend was evident for E. pileatus (R² = 0.71), but not C. velificatus (R² = 0.49). Over a wide range of suspended particulate concentrations (10.6–95.2 mg l^–1), there was no systematic effect of particulates on carbon-specific ingestion rate for any of the three copepod species. However, A. tonsa appeared more adept at grazing in highly turbid water than C. velificatus or E. pileatus.     

Feeding, excretion and egg production by individuals and populations of the marine, planktonic copepods, Acartia spp. and Centropages furcatus

Diel variations in vertical distribution, gut pigment content,ammonium excretion and egg production were investigated foradult females of Acartia erythraea and A.pacifica in the verticallymixed Inland Sea of Japan and Centropages furcatus in the stratified,neritic Gulf of Mexico. Gut pigment content and egg productionrate were maximal at night and ammonium excretion was maximalduring the daytime. Neither A.erythraea nor A.pacifica adultfemales showed an apparent diel migration, but the former werehighly concentrated in the surface layer during the afternoon.In contrast, C.furcatus adult females showed a clear diel migration,residing immediately above the bottom during the daytime andbeing concentrated between 10 and 25 m depth during the nighttime.Individual-based data on gut content and excretion and egg productionrates were combined with vertical-distribution data to calculatepopulation values. In the Inland Sea of Japan, the resultantpattern for Acartia spp. reflected the diel variation in physiologicalrates and even distribution of adult females, except for theafternoon, surface aggregation of A.erythraea. In the Gulf ofMexico, the pattern for C.furcatus reflected largely the dielvariation in each rate process and the heterogeneous distributionof adult females in the water column. Elevated nocturnal feedingactivity of these copepods may be due to an endogenous rhythm.The daytime maximum in ammonium excretion and night-time maximumin egg production rate indicated approximate half-day and daytime lags, respectively, after the intake of food until itsconversion into dissolved excreta and released eggs.     

Feeding rates and productivity of the copepod Acartia bifilosa in a highly turbid estuary; the Gironde (SW France)

Acartia spp. are the dominant copepod species in the Gironde estuary, seaward of the turbidity maximum area. Acartia bifilosa develop a large population in spring and early summer whereas Acartia tonsa appear in late summer. High values and high variability of chlorophyll a/suspended particulate matter ratio are found seaward of the turbidity maximum area. Feeding rates of A. bifilosa were measured by fluorometry. Phytoplankton ingestion was found to be highly variable, between 8 to 80% of copepod carbon body weight. Except for adult females, copepods were heavier in summer than in winter. PB ratios, estimated by the instantaneous growth rate method, varied from 0.03 d^–1 to 0.14 d^–1. The gut contents and P/B ratios of Acartia bifilosa were related to chl a/SPM ratio. From those data, and a few obtained for A. tonsa, it is concluded that only in summer months phytoplankton ingestion is enough to maintain secondary production.     

Feeding preference of the rice stink bug on annual grasses and sedges

Adults of the rice stink bug, Oebalus pugnax (F.), preferred panicles of vasey grass, Paspalum urvillei Steud., over those of 11 species of other grasses and one species of sedge (Cyperus) associated with cultivated rice, Oryza sativa (L.). The number of feeding adults increased approximately 2 times from 1 h post-infestation (PI) to 6 h PI. The number of feeding females was greater than males at 1 h PI but no different at 6 h PI. Both sexes fed significantly (P<0.05) more on O. sativa panicles than on P. urvillei panicles at 6 h PI. Both sexes also fed significantly (P<0.05) more on 28 and 21 cm long O. sativa panicles than on 7 cm long panicles, but no such differences existed in feeding among P. urvillei panicles.

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Résumé Les adultes d'Oebalus pugnax F. ont préféré les panicules de Paspalum urivillei Stend à celles de 11 autres Graminées et 1 Cypéracée associées au riz cultivé, Oryza sativa L. Le nombre d'adultes s'alimentant a cru environ 2 fois de la lère (1P1) à la 6ème (6P1) heure ayant suivi l'infestation. Le nombre de femelles s'alimentant était supérieur à celui des mâles 1 heure après (1P1), mais ne présentait pas de différence 6 h après l'infestation (6P1). Les 2 sexes s'alimentaient significativement plus (P< 0.05) sur les panicules d'O. sativa que sur celles de P. urvillei 6 heures après l'infestation (6P1). Les 2 sexes se sont alimentés significativement plus (P< 0,05) sur des panicules d'O. sativa de 28 et 21 cm que sur ceux de 7 cm, mais aucune différence n'était observée dans l'alimentation sur les panicules de P. urvillei.

     

Fitness consequences for copepods feeding on a red tide dinoflagellate: deciphering the effects of nutritional value, toxicity, and feeding behavior

Phytoplankton exhibit a diversity of morphologies, nutritional values, and potential chemical defenses that could affect the feeding and fitness of zooplankton consumers. However, how phytoplankton traits shape plant–herbivore interactions in the marine plankton is not as well understood as for terrestrial or marine macrophytes and their grazers. The occurrence of blooms of marine dinoflagellates such as Karenia brevis suggests that, for uncertain reasons, grazers are unable to capitalize on, or control, this phytoplankton growth—making these systems appealing for testing mechanisms of grazing deterrence. Using the sympatric copepod Acartia tonsa, we conducted a mixed diet feeding experiment to test whether K. brevis is beneficial, toxic, nutritionally inadequate, or behaviorally rejected as food relative to the palatable and nutritionally adequate phytoplankter Rhodomonas lens. On diets rich in K. brevis, copepods experienced decreased survivorship and decreased egg production per female, but the percentage of eggs that hatched was unaffected. Although copepods showed a 6–17% preference for R. lens over K. brevis on some mixed diets, overall high ingestion rates eliminated the possibility that reduced copepod fitness was caused by copepods avoiding K. brevis, leaving nutritional inadequacy and toxicity as remaining hypotheses. Because egg production was dependent on the amount of R. lens consumed regardless of the amount of K. brevis eaten, there was no evidence that fitness costs were caused by K. brevis toxicity. Copepods limited to K. brevis ate 480% as much as those fed only R. lens, suggesting that copepods attempted to compensate for low food quality with increased quantity ingested. Our results indicate that K. brevis is a poor food for A. tonsa, probably due to nutritional inadequacy rather than toxicity, which could affect bloom dynamics in the Gulf of Mexico where these species co-occur.