Can cryptophyte abundance trigger toxic Karlodinium veneficum blooms in eutrophic estuaries?

Karlodinium veneficum is a common member of the phytoplankton in coastal ecosystems, usually present at relatively low cell abundance (10² to 10³ mL⁻¹), but capable of forming blooms of 10⁴ to 10⁵ cells mL⁻¹ under appropriate conditions. We present evidence consistent with the hypothesis that prey abundance, particularly the abundance of nano-planktonic cryptophytes, is a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments. K. veneficum is known to increase growth rate 2- to 3-fold in culture through mixotrophic nutrition, but the role of feeding in bloom formation has not been directly examined. We find that toxic K. veneficum blooms are correlated with cryptophytes abundance changes. We find a wide range of mixotrophic feeding capabilities (0–4 prey per predator per day) among genetically distinct strains of K. veneficum when fed a common prey. Finally, we find that toxic K. veneficum is capable of feeding on a wide range of cryptophyte species varying in size (31–421 μm³ per cell) and phylogenetic affinity, although ingestion rates of different prey vary significantly. While abiotic conditions (e.g. nutrients and advection) are an important aspect of K. veneficum bloom formation in eutrophic environments, our results reinforce the need for a broader view of conditions leading to toxic K. veneficum blooms including biotic factors such as prey availability.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.     

The interplay between host toxins and parasitism by Amoebophrya

The parasitic dinoflagellate Amoebophrya sp. ex Karlodinium veneficum was used to test two hypotheses: (1) infection of cells decreases with increasing host toxicity and (2) parasitism causes the catabolism of host toxin. To test the first hypothesis, host strains differing in toxin content were inoculated with dinospores of Amoebophrya sp. derived from infected cultures of toxic and non-toxic K. veneficum, with resulting infections assessed following 24-h incubations. Contrary to expectations, infection of K. veneficum by Amoebophrya sp. was positively correlated with host toxicity. To examine the second hypothesis, synchronous infection with >80% of cells being parasitized was induced using a toxic strain of K. veneficum, and total toxin concentration (intracellular plus extracellular levels of KmTX1) was followed over the 3-day infection cycle. Toxin content ml⁻¹ increased with growth of K. veneficum in uninfected control cultures, but declined in infected cultures as the parasite completed its life cycle. On a cellular basis, toxin content of infected and uninfected cultures differed little during the experiment, suggesting that the parasite does not actively catabolise host toxin. Rather, infection appears to promote degradation of toxins via death of host cells and subsequent bacterial activity. Results indicate that Amoebophrya sp. ex K. veneficum has greater potential to impact toxic strains relative to non-toxic host strains in natural systems. Thus, Amoebophrya sp. ex. K. veneficum may limit the occurrence of toxic K. veneficum blooms in marine and estuarine environments, while simultaneously functioning as a pathway for dissipation of host toxin.     

Modulation of polyunsaturated fatty acids in mixotrophic Karlodinium veneficum (Dinophyceae) and its prey,Storeatula major (Cryptophyceae)1

We examined whether fatty acid (FA) composition changed when Karlodinium veneficum (D. Ballantine) J. Larsen (Dinophyceae) was grown phototrophically or mixotrophically on Storeatula major Butcher ex D. R. A. Hill (Cryptophyceae). We hypothesized that the FA composition of mixotrophic K. veneficum would not change relative to the FA composition of phototrophic K. veneficum. As in other phototrophic dinoflagellates, octadecapentaenoic acid (18:5n3) represented 9% to 20% of total FA in K. veneficum and was enriched within chloroplast‐associated galactolipid classes. The 18:5n3 content showed a highly significant positive correlation (r² = 0.95) with chl a content and a highly significant negative correlation with growth rate (r² = 0.88). A previously undescribed chloroplast galactolipid molecular species, digalactosyldiacylglycerol (DGDG; 18:5n3/18:5n3), was a dominant structural lipid in K. veneficum. Docosahexaenoic acid (22:6n3) represented 14% to 19% of total K. veneficum FA and was enriched within phospholipids. In the prey S. major, 18:5n3 was not present, but octadecatetraenoic acid (18:4n3) and α‐linolenic acid (18:3n3) represented approximately 50% of total FA and were enriched within chloroplast‐associated galactolipid classes. Eicosapentaenoic acid (20:5n3) and 22:6n3 represented approximately 18% of total FA in S. major and were enriched within phospholipids. The FA profile of mixotrophic K. veneficum, compared to phototrophic K. veneficum, showed elevated levels of 18:3n3, 18:4n3, and 20:5n3, and lower but persistent levels of 18:5n3. Production to ingestion (P:I) ratios >1 for major polyunsaturated fatty acids (PUFAs) indicated that direct assimilation from prey under balanced growth could not support rates of PUFA production in mixotrophic K. veneficum. These data suggest that the plastid plays a continuing and essential role in lipid metabolism during mixotrophic growth.     

Mixotrophy in the sand-dwelling dinoflagellate Thecadinium kofoidii

Thecadinium kofoidii is a marine sand-dwelling dinoflagellate that sometimes forms dense blooms. This species was previously thought to be an exclusively autotrophic dinoflagellate, and its mixotrophic ability has not been explored yet. By investigating its ecophysiology, its trophic mode should be revealed. We explored the mixotrophic ability of T. kofoidii by examining its protoplasm under light and transmission electron microscopes with diverse algal prey species. Furthermore, the feeding mechanism of T. kofoidii and prey species on which it feeds were investigated. In addition, the growth and ingestion rates of T. kofoidii as a function of prey concentration were determined when feeding on the benthic cryptophyte Rhodomonas salina. Thecadinium kofoidii was able to feed on R. salina and the dinoflagellate Symbiodinium voratum, which had equivalent spherical diameters (ESDs) ≤ 10.1?µm, while it did not feed on the benthic dinoflagellates Levanderina fissa, Prorocentrum concavum or Ostreopsis cf. ovata, which had ESDs ≥ 15?µm. Thecadinium kofoidii fed on the edible prey cells using the peduncle. The maximum ingestion rate of T. kofoidii on R. salina was 1.3 cells predator^?1 d^?1. However, feeding on R. salina did not significantly increase the growth rate of T. kofoidii. The low ingestion rate of T. kofoidii on R. salina may have partially resulted in the lack of significant increase in its growth rate due to mixotrophy. The present study discovered predator–prey relationships between T. kofoidii and R. salina and S. voratum, which may change our view of the energy flow and carbon cycling in marine benthic food webs.     

Cylindrospermopsin production and release by the potentially invasive cyanobacterium Aphanizomenon ovalisporum under temperature and light gradients

The growth rates, production and release of the potent cytotoxin cylindrospermopsin (CYN) were studied in batch and semi-continuous cultures of Aphanizomenon ovalisporum (Cyanobacteria; Nostocaceae) strains UAM 289 and UAM 290 from Spain, over a gradient of temperatures (10–40 °C) and irradiances (15–340 μE m⁻² s⁻¹). This species grew in temperatures ranging from 15 °C to 35 °C as well as under all irradiances assayed. The growth rates ranged from 0.08 d⁻¹ to 0.35 d⁻¹, and the maximum growth was recorded above 30 °C and at 60 μE m⁻² s⁻¹. CYN was produced under all conditions where net growth occurred. Total CYN reached up to 6.4 μg mg⁻¹ dry weight, 2.4 μg mm⁻³ biovolume, 190.6 fg cell⁻¹ and 0.5 μg μg⁻¹ chlorophyll a. Although CYN concentrations varied only 1.9-fold within the 15–30 °C range, a drastic 25-fold decrease was observed at 35 °C. The irradiance induced up to 4-fold variations, with maximum total CYN measured at 60 μE m⁻² s⁻¹. An elevated extracellular CYN share ranging from 20% to 35% was observed during the exponential growth phase in most experiments, with extreme temperatures (15 and 35 °C) being related to the highest release (63% and 58%, respectively) and without remarkable influence of irradiance. Growth did not have a direct influence on either CYN production or release throughout the entire range of experimental conditions. Our study demonstrates a strong and stable production and release of CYN by A. ovalisporum along field-realistic gradients of temperature and light, thus becoming a predictive tool useful for the management of water bodies potentially affected by this ecologically plastic cyanobacterium.     

An unusual bloom of the dinoflagellate Akashiwo sanguinea off the central Oregon, USA, coast in autumn 2009

An algal bloom caused by the dinoflagellate Akashiwo sanguinea was observed in October–November 2009 along the central Oregon coast (44.6°N), off Newport, Oregon, U.S.A. In this paper, the conditions are described which led to the development and demise of this bloom. The bloom was observed for 1 month from 5-October until 4-November with the peak of abundance on 19-October (347,615 cells L⁻¹). The A. sanguinea bloom followed September blooms of the diatoms Pseudo-nitzschia spp, Chaetoceros debilis, and the dinoflagellate Prorocentrum gracile. The bloom occurred when nitrate and silicate concentrations were <2 μM and <8 μM, respectively, and when the water column was stratified. This A. sanguinea dinoflagellate bloom event was closely related to the anomalous upwelling conditions in 2009: upwelling ceased early, at the end of August, whereas a normal upwelling continues into early October. This relaxation extended to near the end of September as a prolonged downwelling event, but then active upwelling reappeared in October and November. The explanation for the occurrence of the A. sanguinea bloom in October may be related to a combination of a prior diatom bloom, a stratified water column with low nutrient concentration in September, and an active upwelling event in October. As for the ultimate source of the cells, the hypothesis is that the seed stock for the A sanguinea bloom off Oregon was southward transport of cells from the Washington coast where a massive bloom of A. sanguinea was first observed in September 2009.     

Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal sea of Japan

Noxious red tides of the dinoflagellate Cochlodinium polykrikoides tend to be long lasting and cause mass mortalities of cultured and natural fish and invertebrates along the western coast of Japan and the southern coast of Korea. In order to assess the tolerance of C. polykrikoides to attack by algicidal bacteria, the effects of algicidal bacteria strains on the growth of three C. polykrikoides strains were examined in laboratory culture experiments. Algicidal bacteria used were two strains of Cytophaga (J18/M01 and AA8-2, direct attack type and wide prey range), three strains of Alteromonas (S, K, D) and one strain of Pseudoalteromonas (R, indirect attack type), which were all isolated by using Chattonella antiqua as a prey organism. Neither Cytophaga strain showed any algicidal activity. In the cases of Alteromonas and Pseudoalteromonas, some cultures of C. polykrikoides were killed, but at least 10 days or more were required for the death of this dinoflagellate. C. polykrikoides survived in the presence of algicidal bacteria in concentrations up to 10⁶–10⁷ cells ml⁻¹, which is enough for other red tide microalgae to be killed. On the contrary, the algicidal effects of bacteria on C. antiqua were detected clearly within a few days. These results imply that C. polykrikoides is resistant to the six algicidal bacteria examined, which may reflect the capacity for mixotrophy. This resistance of C. polykrikoides to algicidal bacteria could provide a selective advantage for survival compared to other microalgae susceptible to attack by algicidal bacteria and hence prolong red tides caused by this harmful dinoflagellate.     

CHARACTERIZATION OF NW MEDITERRANEAN KARLODINIUM SPP. (DINOPHYCEAE) STRAINS USING MORPHOLOGICAL,MOLECULAR, CHEMICAL,AND PHYSIOLOGICAL METHODOLOGIES1

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug‐like structures in amphiesma and a possible layer of semi‐opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low‐ (K. veneficum) and high‐light (K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates.     

Use of the dinoflagellate <Emphasis Type="Italic">Karlodinium veneficum</Emphasis> as a sustainable source of biodiesel production

Microalgae are microscopic heterotrophic–autotrophic photosynthesizing organisms with enormous potential as a source of biofuel. Dinoflagellates, a class of microalgae, contain large amounts of high-quality lipids, the principal component of fatty acid methyl esters. The biotic characteristics of the dinoflagellate species Karlodinium veneficum include a growth rate of 0.14 day⁻¹, a wet biomass of 16.4 g/L, a growth period of approximately 30 days, and an approximate 97% increase in fatty acid content during the transition from exponential phase to stationary phase. These parameters make K. veneficum a suitable choice as a bioresource for biodiesel production. Similarly, two other species were also determined to be appropriate for biodiesel production: the Dinophyceae Alexandrium andersoni and the Raphidophyte Heterosigma akashiwo.     

Growth,Grazing, and Starvation Survival in Three Heterotrophic Dinoflagellate Species

To assess the effects of fluctuating prey availability on predator population dynamics and grazing impact on phytoplankton, we measured growth and grazing rates of three heterotrophic dinoflagellate species—Oxyrrhis marina, Gyrodinium dominans and Gyrodinium spirale—before and after depriving them of phytoplankton prey. All three dinoflagellate species survived long periods (> 10 d) without algal prey, coincident with decreases in predator abundance and cell size. After 1–3 wks, starvation led to a 17–57% decrease in predator cell volume and some cells became deformed and transparent. When re‐exposed to phytoplankton prey, heterotrophs ingested prey within minutes and increased cell volumes by 4–17%. At an equivalent prey concentration, continuously fed predators had ~2‐fold higher specific growth rates (0.18 to 0.55 d^?1) than after starvation (?0.16 to 0.25 d^?1). Maximum specific predator growth rates would be achievable only after a time lag of at least 3 d. A delay in predator growth poststarvation delays predator‐induced phytoplankton mortality when prey re‐emerges at the onset of a bloom event or in patchy prey distributions. These altered predator‐prey population dynamics have implications for the formation of phytoplankton blooms, trophic transfer rates, and potential export of carbon.     

Marine microalgae attack and feed on metazoans

Free-living microalgae from the dinoflagellate genus Karlodinium are known to form massive blooms in eutrophic coastal waters worldwide and are often associated with fish kills. Natural bloom populations, recently shown to consist of the two mixotrophic and toxic species Karlodinium armiger and Karlodinium veneficum have caused fast paralysis and mortality of finfish and copepods in the laboratory, and have been associated with reduced metazooplankton biomass in-situ. Here we show that a strain of K. armiger (K-0688) immobilises the common marine copepod Acartia tonsa in a density-dependent manner and collectively ingests the grazer to promote its own growth rate. In contrast, four strains of K. veneficum did not attack or affect the motility and survival of the copepods. Copepod immobilisation by the K. armiger strain was fast (within 15 min) and caused by attacks of swarming cells, likely through the transfer and action of a highly potent but uncharacterised neurotoxin. The copepods grazed and reproduced on a diet of K. armiger at densities below 1000, cells ml⁻¹, but above 3500 cells ml⁻¹ the mixotrophic dinoflagellates immobilised, fed on and killed the copepods. Switching the trophic role of the microalgae from prey to predator of copepods couples population growth to reduced grazing pressure, promoting the persistence of blooms at high densities. K. armiger also fed on three other metazoan organisms offered, suggesting that active predation by mixotrophic dinoflagellates may be directly involved in causing mortalities at several trophic levels in the marine food web.     

Potential impacts of brown tide, Aureococcus anophagefferens, on juvenile hard clams, Mercenaria mercenaria, in the Coastal Bays of Maryland, USA

The rate of growth of juvenile hard clams, Mercenaria mercenaria, was studied in the Coastal Bays of Maryland during an outbreak of the brown tide, Aureococcus anophagefferens. Brown tide dominated the plankton community during the month of June 2002, with cell densities at several sites reaching category 3 (>200,000 cells ml⁻¹) levels. Temperatures during the bloom were 18.6–27.5 °C. Nutrient conditions preceding and during the bloom were conducive for the proliferation of A. anophagefferens: while inorganic nitrogen and phosphorus were <1 μg at N or P l⁻¹, urea was elevated during bloom development. Organic nitrogen, phosphorus and carbon were in the range of levels observed in previous brown tide blooms and increased following the collapse of the bloom. Growth rates of juvenile clams were significantly lower during the period of the brown tide bloom than following its collapse. Growth rates of M. mercenaria were found to be negatively impacted at brown tide densities as low as 20,000 cells ml⁻¹, or category 1 levels. The low growth rates of M. mercenaria could not be explained by temperature, as the lowest growth rates were found when water temperatures were at levels previously found to be optimal for growth.     

Brominated aliphatic hydrocarbons and sterols from the sponge Xestospongia testudinaria with their bioactivities

Four brominated aliphatic hydrocarbons (1–4), including a novel brominated ene-tetrahydrofuran named as mutafuran H (1), and five sterols (5–9) were isolated from the South China Sea sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of NMR (¹H, ¹³C NMR, HSQC, HMBC, ¹H–¹H COSY, and NOESY), MS, and optical rotation analysis. Known compounds were identified by comparison of their NMR data with those reported in the literature. Compounds 1–4, and 6–9 were evaluated for their toxicity against Artemia salina larvae, and anti-acetylcholinesterase activity.     

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliella salina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO₄-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 10⁶ L⁻¹ with typical densities of 1.5–4.5 × 10⁶ L⁻¹. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L⁻¹ within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO₃-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO₄-P applications (4.0 and 8.0 mg L⁻¹) at 60 ppt salinity and 23 °C. The lower PO₄-P applications (0.6–2.0 mg L⁻¹) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.     

Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies

The Delaware Inland Bays (DIB) have experienced harmful algal blooms of dinoflagellates and raphidophytes in recent years. We used quantitative polymerase chain reaction (QPCR) techniques to investigate the community dynamics of three DIB dinoflagellates (Karlodinium veneficum, Gyrodinium instriatum, and Prorocentrum minimum) and one raphidophyte (Heterosigma akashiwo) at a single site in the DIB (IR-32) in summer 2006 relative to salinity, temperature and nutrient concentrations. We also carried out complementary laboratory culture studies. New primers and probes were developed and validated for the 18S rRNA genes in the three dinoflagellates. K. veneficum, H. akashiwo, and G. instriatum were present in almost all samples throughout the summer of 2006. In contrast, P. minimum was undetectable in late June through September, when temperatures ranged from 20 to 30 °C (average 25.7 °C). Dissolved nutrients ranged from 0.1 to 2.8 μM PO₄³⁻ (median = 0.3 μM), 0.7–30.2 μM NO_x (median = 12.9 μM), and 0–19.4 μM NH₄⁺ (median = 0.7 μM). Dissolved N:P ratios covered a wide range from 2.6 to 177, with a median of 40. There was considerable variability in occurrence of the four species versus nutrients, but in general P. minimum and H. akashiwo were most abundant at higher (>40) N:P ratios and dissolved nitrogen concentrations, while K. veneficum and G. instriatum were most abundant at low dissolved N:P ratios (<20) and dissolved nitrogen concentrations < 10 μM. The semi-continuous laboratory competition experiment used mixed cultures of K. veneficum, P. minimum, and H. akashiwo grown at dissolved N:P ratios of 5, 16, and 25. At an N:P of 16 and 25 P. minimum was the dominant alga at the end of the experiment, even at a temperature that was much higher than that at which this alga was found to bloom in the field (27 °C). P. minimum and H. akashiwo had highest densities in the N:P of 25. K. veneficum grew equally well at all three N:P ratios, and was co-dominant at times at an N:P of 5. H. akashiwo had the lowest densities of the three algae in the laboratory experiment. Laboratory and field results showed both interesting similarities and significant differences in the influences of important environmental factors on competition between these harmful algal species, suggesting the need for more work to fully understand HAB dynamics in the DIB.     

Investigation of Chondrus crispus as a potential source of new antifouling agents

The search for environment-friendly and non-toxic antifouling (AF) paint components has led to the investigation of natural products from seaweeds. The defence metabolites used by algae to deter unwanted epibiosis have potential for harnessing and use in AF applications. Crude algal extracts may provide a suitable mixture of compounds with AF potency. Crude ethanol extracts of the macroalgae Chondrus crispus (Rhodophyceae), from both dried and fresh sources were tested and compared using bioassays based on five marine bacterial strains, five phytoplankton strains and two macroalgae to assess the AF efficacy. Dried extract from the algae had a lower minimum inhibitory concentration at 25 μg mL⁻¹ against the growth of bacteria and phytoplankton species than that from the fresh source. Macroalgae tests indicated that the extracts had an anti-germination activity 25–50 μg mL⁻¹ against both Undaria pinnatifida and Ulva intestinalis spores. A field trial of AF paint incorporating crude extract indicated an initial AF potency lasting six weeks.     

Grazing impact of microzooplankton on a diatom bloom in a mesocosm as estimated by pigment-specific dilution technique

To investigate the impact of microzooplankton grazing on phytoplankton bloom in coastal waters, an enclosure experiment was conducted in Saanich Inlet, Canada during the summer of 1996. Daily changes in the microzooplankton grazing rate on each phytoplankton group were investigated with the growth rates of each phytoplankton group from the beginning toward the end of bloom using the dilution technique with high-performance liquid chromatography (HPLC). On Day 1 when nitrate and iron were artificially added, chlorophyll a concentration was relatively low (4.3 μg l⁻¹) and 19′-hexanoyloxyfucoxanthin-containing prymnesiophytes were predominant in the chlorophyll biomass. However, both the synthetic rates and concentrations of 19′-hexanoyloxyfucoxanthin declined before bloom, suggesting that 19′-hexanoyloxyfucoxanthin-containing prymnesiophytes weakened. Chlorophyll a concentration peaked at 23 μg l⁻¹ on Day 4 and the bloom consisted of the small chain-forming diatoms Chaetoceros spp. (4 μm in cell diameter). Diatoms were secondary constituents in the chlorophyll biomass at the beginning of the experiment, and the growth rates of diatoms (fucoxanthin) were consistently high (>0.5 d⁻¹) until Day 3. Microzooplankton grazing rates on each phytoplankton group remarkably increased except on alloxanthin-containing cryptophytes after the nutrient enrichments, and peaked with >0.6 d⁻¹ on Day 3, indicating that >45% of the standing stock of each phytoplankton group was removed per day. Both the growth and mortality rates of alloxanthin-containing cryptophytes were relatively high (>1 and >0.5 d⁻¹, respectively) until the bloom, suggesting that a homeostatic mechanism might exist between predators and their prey. Overall, microzooplankton grazing showed a rapid response to the increase in phytoplankton abundance after the nutrient enrichments, and affected the magnitude of the bloom significantly. High grazing activity of microzooplankton contributed to an increase in the abundance of heterotrophic dinoflagellates with 7-24 μm in cell size, the fraction of large-sized (>10 μm) chlorophyll a, and stimulated the growth of larger-sized ciliates after the bloom.     

Bioenergetics and growth in the ctenophore <Emphasis Type="Italic">Pleurobrachia pileus</Emphasis>

Knowledge of how energetic parameters relate to fluctuating factors in the natural habitat is necessary when evaluating the role of gelatinous zooplankton in the carbon flow of coastal waters. In laboratory experiments, we assessed feeding, respiration and growth of the ctenophore, Pleurobrachia pileus, and constructed carbon budgets. Clearance rates (F, l d⁻¹) of laboratory-reared Acartia tonsa as prey increased as a function of ctenophore polar length (L, mm) as F = 0.17L ^1.9. For ctenophores larger than about 11 mm, clearance rate was depressed in containers of 30–50 l volume. Clearance rates on field-collected prey were highest on the copepod, Centropages typicus, intermediate on the cladoceran, Evadne nordmanni and low on the copepods, Acartia clausi and Temora longicornis. Specific growth rates of 8–10 mm P. pileus increased with increasing prey concentrations to a maximum of 0.09 d⁻¹ attained at prey carbon densities of 40 and 100 μg C l⁻¹ of Artemia salina and A. tonsa, respectively. Weight-specific respiration rates increased hyperbolically with prey concentration. From experiments in which growth, ingestion and respiration were measured simultaneously, a carbon budget was constructed for individuals growing at maximum rates; from the measured parameters, the assimilation efficiency and net growth efficiency were estimated to be 22 and 37%, respectively. We conclude that the predation rates of P. pileus depend on ctenophore size, prey species, prey density and experimental container volume. Because the specific growth rates, respiration, assimilation and net growth efficiencies all were affected by food availability, knowledge of the ambient prey field is critical when evaluating the role of P. pileus in the carbon flow in coastal waters.