Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

Red tide blooms of Cochlodinium polykrikoides in a coastal cove

Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from <100 cells L⁻¹ at the onset of the bloom and reached a maximum density exceeding 3.4 × 10⁶ cells L⁻¹ during the summer of 1981. The bloom was mainly restricted to the mid to inner region of this shallow cove with greatest concentrations localized in surface waters of the southwestern region during summer/fall periods of both years. Highly motile cells consisting of single, double and multiple cell zooids were found as chains of 4 and 8 cells restricted to the late August/September periods. The highest cell densities occurred during periods when annual temperatures were between 19 and 28 °C and salinities between 25 and 30. A major nutrient source for the cove was Crying Brook, located at the innermost region at the head of the cove. Inorganic nitrogen (NH₃ and NO₂ + NO₃) from the brook was continually detectable throughout the study with maximum values of 57.5 and 82.5 μmol L⁻¹, respectively. Phosphate (PO₄-P) was always present in the source waters and rarely <0.5 μmol L⁻¹; silicate always exceeded 30 μmol L⁻¹ with maximum concentrations reaching 226 μmol L⁻¹. Chlorophyll a and ATP concentrations during the blooms varied directly with cell densities. Maximum Chl a levels were 218 mg m⁻³ and ATP-carbon was >20 g C m⁻³. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m⁻³ d⁻¹. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d⁻¹. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.     

Anatomy of a red tide bloom off the southwest coast of Florida

A massive outbreak of Karenia brevis that had been ongoing for several months along the southwestern coast of Florida was sampled in early September 2005 off Sanibel Island to assess the utility of bio-optical features and ataxonomic analysis (quantification of eukaryotic and cyanobacterial picoplankton) by flow cytometry in monitoring red tide blooms. Sea-surface sampling followed aircraft visual location of discolored water. Within the most concentrated area of the bloom, chlorophyll a values exceeded 500 μg l⁻¹, and concentrations of nitrate (0.3 μM ± 0.0) and ammonium (<0.2 μM) were depleted compared to high concentrations of total dissolved nitrogen, total dissolved phosphorus, and soluble reactive phosphorus (141 ± 34 μM, 16.5 ± 2.5 μM, and 6.44 ± 0.57 μM, respectively). Low water clarity in the bloom (Secchi depth transparency 0.3 m, K_d estimated at 4.83 m⁻¹) was strongly influenced by attenuation from dinoflagellates as well as chromophoric dissolved organic matter (CDOM). The fact that the K. brevis bloom occurred in lower-salinity (30 psu), high-nutrient waters implicates riverine transport of land-based nutrients as a source of nutrient supplies that fueled or sustained the bloom. Throughout ongoing efforts to advance modeling and technological capabilities that presently lack reliable predictive capability, bio-optical remote sensing via aerial flyovers along with in-water sensor data can continue to provide accurate coverage of relatively large temporal and spatial features. Flow cytometry can provide conservative (because of some cell lysis), rapid, near-real-time validation of bloom components. The concentration and position of the organisms, along with water mass scalars, can also help to diagnose factors promoting K. brevis bloom development and dispersion.     

Impacts and potential effects due to Prorocentrum minimum blooms in Chesapeake Bay

The dinoflagellate Prorocentrum minimum (P. minimum) can be found in all seasons and over a broad range of habitat conditions in the Chesapeake Bay and its tributaries. Blooms (>3000 cells ml⁻¹), locally referred to as ‘mahagony tides’, were restricted to salinities of 4.5–12.8 psu, water temperatures of 12–28 °C, and occurred most frequently in April and May. P. minimum blooms have been detected at routine water quality monitoring stations located in the main channel of the Bay and tidal tributaries. Nearshore investigations of bloom events, however, have accounted for the majority of events recorded in excess of 10⁵ cells ml⁻¹. Mahogany tides were associated with widespread harmful impacts including anoxic/hypoxic events, finfish kills, aquaculture shellfish kills and submerged aquatic vegetation losses. We summarize the state of knowledge regarding physical and chemical factors related to P. minimum blooms, their abundance, distribution and frequency, and ecological effects in Chesapeake Bay.     

Occurrence and toxicity of an Anabaena bloom in a tropical reservoir (Southeast Brazil)

Potentially toxic cyanobacterial blooms are becoming common in the Brazilian reservoirs in all regions of the country. During October 2004, a dense bloom of cyanobacteria occurred in the Monjolinho Reservoir (São Carlos, São Paulo State, Brazil) and a significant amount of cyanobacterial material accumulated on the water surface. Phytoplankton analysis showed that the main species in this bloom were Anabaena circinalis and Anabaena spiroides. Cladoceran (Ceriodaphnia dubia and Ceriodaphnia silvestrii) and mouse bioassays were performed to detect toxic products in extracts of the natural samples collected at the three different dates during in short period. To prepare the extracts, freeze-dried cells were dispersed in distilled water and subjected to repeated freeze/thaw cycles and sonication and centrifuging processes. Crude extracts were toxic both to cladocerans (LC₅₀ 94–406 mg freeze-dried cells L⁻¹) and mice (indicative LD₅₀ 297–445 mg freeze-dried cells kg⁻¹) and the toxicity of the bloom increased for cladocerans during the occurrence of the bloom. Toxin analysis by ELISA revealed that microcystin (MC) was found in the water of the reservoir (concentrations ranging from 28 to 45 μg L⁻¹). In addition, microcystin was also found in freeze-dried cyanobacteria cells with concentrations ranging from 138 to 223 μg g⁻¹. On the other hand, neurotoxins (saxitoxin and gonyautoxin) were not detected in any of the natural samples by HPLC. Signs of toxicity in mice did not indicate whether the bloom samples were predominantly hepatotoxic or neurotoxic. It is known that natural Anabaena blooms can contain other toxic compounds besides microcystins and neurotoxins such as lipopolysaccharides or other toxins not identified or known. Methods of detecting cyanotoxins used in this study were insufficient to clarify the toxicological features of Anabaena bloom and indicated that other methods should be investigated.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Prorocentrum minimum blooms: potential impacts on dissolved oxygen and Chesapeake Bay oyster settlement and growth

The cosmopolitan dinoflagellate Prorocentrum minimum is a recurrent bloom forming species in the Chesapeake Bay and its tributaries, generally observed at its highest levels in late spring and summer. Laboratory studies were conducted to assess potential bloom impacts on diel oxygen concentrations in shallow littoral zones as well as settlement success and post-set growth of the eastern oyster Crassostrea virginica. Using light–dark and dark cultures and periodic diel sub-sampling, bloom levels of P. minimum produced supersaturated oxygen levels at the end of each day while darkened cultures were typified by rapid decreases in dissolved oxygen (DO) (1.1–1.3 mg L⁻¹ h⁻¹) to hypoxic and anoxic levels within 4 days. These data suggest shallow, poorly flushed systems and the biota in them will experience rapid and large diel variations in oxygen, implying recurrent P. minimum blooms need be considered as short-term oxygen stressors for Bay oyster spat and other living resources. Direct effects of P. minimum impacts on oysters were not as expected or previously reported. In one experiment, pre-bloom isolates of P. minimum were grown and then exposed to polyvinyl chloride (PVC) settlement plates to see whether dinoflagellate preconditioning of the hard substrate might affect oyster sets. No differences were noted between set on the PVC with P. minimum exposure to set recorded with filtered seawater, Instant Ocean^®, or Isochrysis. In the second oyster experiment, spat on PVC plates were exposed to field collected P. minimum blooms and a commercial mixture of several other food types including Isochrysis. Oyster growth was significantly higher in P. minimum exposures than noted in the commercial mix. These results, compared to results with other isolates from the same region, indicate substantial positive impact from some of the P. minimum blooms of the area while others separated in space, time, or nutrient status could severely curtail oyster success through toxin production induced by nutrient limitation.     

Ecophysiology of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae) in Moreton Bay, Australia

Large blooms of the marine cyanobacterium Lyngbya majuscula in Moreton Bay, Australia (27°05′S, 153°08′E) have been re-occurring for several years. A bloom was studied in Deception Bay (Northern Moreton Bay) in detail over the period January–March 2000. In situ data loggers and field sampling characterised various environmental parameters before and during the L. majuscula bloom. Various ecophysiological experiments were conducted on L. majuscula collected in the field and transported to the laboratory, including short-term (2 h) ¹⁴C incorporation rates and long-term (7 days) pulse amplitude modulated (PAM) fluorometry assessments of photosynthetic capacity. The effects of L. majuscula on various seagrasses in the bloom region were also assessed with repeated biomass sampling. The bloom commenced in January 2000 following usual December rainfall events, water temperatures in excess of 24 °C and high light conditions. This bloom expanded rapidly from 0 to a maximum extent of 8 km² over 55 days with an average biomass of 210 g_dw⁻¹ m⁻² in late February, followed by a rapid decline in early April. Seagrass biomass, especially Syringodium isoetifolium, was found to decline in areas of dense L. majuscula accumulation. Dissolved and total nutrient concentrations did not differ significantly (P > 0.05) preceding or during the bloom. However, water samples from creeks discharging into the study region indicated elevated concentrations of total iron (2.7–80.6 μM) and dissolved organic carbon (2.5–24.7 mg L⁻¹), associated with low pH values (3.8–6.7). ¹⁴C incorporation rates by L. majuscula were significantly (P < 0.05) elevated by additions of iron (5 μM Fe), an organic chelator, ethylenediaminetetra-acetic acid (5 μM EDTA) and phosphorus (5 μM PO₄⁻³). Photosynthetic capacity measured with PAM fluorometry was also stimulated by various nutrient additions, but not significantly (P > 0.05). These results suggest that the L. majuscula bloom may have been stimulated by bioavailable iron, perhaps complexed by dissolved organic carbon. The rapid bloom expansion observed may then have been sustained by additional inputs of nutrients (N and P) and iron through sediment efflux, stimulated by redox changes due to decomposing L. majuscula mats.     

Paralytic shellfish toxins in zooplankton, mussels, lobsters and caged Atlantic salmon, Salmo salar, during a bloom of Alexandrium fundyense off Grand Manan Island, in the Bay of Fundy

Substantial mortalities of Atlantic salmon (Salmo salar) at two aquaculture sites in Long Island Sound, off Grand Manan Island, Bay of Fundy (BoF) (New Brunswick, Canada) in September 2003, were associated with a bloom of Alexandrium fundyense (>3 × 10⁵ cells L⁻¹), a dinoflagellate alga that produces toxins which cause paralytic shellfish poisoning (PSP). Cells of A. fundyense collected from surface waters while fish were dying had total paralytic shellfish (PS) toxin concentrations of 70.6 pg STX equiv. (saxitoxin equivalents) cell⁻¹ and PS toxin profiles rich in carbamate toxins (78.2%). The zooplankton sampled contained PS toxins (63.1 pg STX equiv. g⁻¹ wet wt) and the toxin profile matched that of A. fundyense cells.Mean PS toxin levels were low (<4 μg STX equiv. 100 g⁻¹ wet wt) in stomach, gill and muscle tissues of moribund salmon, suggesting that PS toxins are very lethal to salmon.The PS toxin concentrations in blue mussels (Mytilus edulis) growing on the salmon cages (37; 526 μg STX equiv. 100 g⁻¹ wet wt) were the highest recorded to date from this region. Their PS toxin profiles showed enhanced carbamate contents (85.5%) compared with that found in A. fundyense. Blue mussels collected from an adjacent Canadian Food Inspection Agency (CFIA) monitoring site in Grand Manan had PS toxin concentrations of 4214 and 150 μg STX equiv. 100 g⁻¹ wet wt in late September and December, respectively, well above the regulatory limit (RL), and horse mussels (Modiolus modiolus) collected in late September had PS toxin concentrations of 2357 μg STX equiv. 100 g⁻¹ wet wt. Detoxification under laboratory conditions suggested that blue mussels may require up to 19 weeks for elimination below RL when they accumulate these high concentrations of PS toxins. This depuration period may be shorter in the field.PS toxin levels above RL were detected in hepatopancreatic tissues of lobster (Homarus americanus), with lower levels (<16 μg STX equiv. 100 g⁻¹ wet wt) in tail muscle and gills.These results illustrate the movement of PS toxins through the marine food chain following an A. fundyense bloom in the BoF, and support earlier studies suggesting that kills from the region of zooplanktivorous fish, such as herring (Clupea harengus harengus), can be attributed to blooms of A. fundyense. This is the first reported incident of PSP associated with mortalities of caged Atlantic salmon in the BoF. Analyses of muscle tissues and viscera from the affected salmon indicated that any portion would not be a health hazard if consumed.     

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.     

Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA

We report on the emergence of Cochlodinium polykrikoides blooms in the Peconic Estuary and Shinnecock Bay, NY, USA, during 2002–2006. Blooms occurred during late summer when temperatures and salinities ranged from 20 to 25 °C and 22 to 30 ppt, respectively. Bloom patches achieved cell densities exceeding 10⁵ ml⁻¹ and chlorophyll a levels exceeding 100 μg l⁻¹, while background bloom densities were typically 10³–10⁴ cells ml⁻¹. Light, scanning electron and ultrathin-section transmission electron microscopy suggested that cells isolated from blooms displayed characteristics of C. polykrikoides and provide the first clear documentation of the fine structure for this species. Sequencing of a hypervariable region of the large subunit rDNA confirmed this finding, displaying 100% similarity to other North American C. polykrikoides strains, but a lower similarity to strains from Southeast Asia (88–90%). Bioassay experiments demonstrated that 24 h exposure to bloom waters (>5 × 10⁴ cells ml⁻¹) killed 100% of multiple fish species (1-week-old Cyprinodon variegates, adult Fundulus majalis, adult Menidia menidia) and 80% of adult Fundulus heteroclitus. Microscopic evaluation of the gills of moribund fish revealed epithelial proliferation with focal areas of fusion of gill lamellae, suggesting impairment of gill function (e.g. respiration, nitrogen excretion, ion balance). Lower fish mortality was observed at intermediate C. polykrikoides densities (10³–10⁴ cells ml⁻¹), while fish survived for 48 h at cell densities below 1 × 10³ cells ml⁻¹. The inability of frozen and thawed-, or filtered (0.2 μm)-bloom water to cause fish mortality suggested that the thick polysaccharide layer associated with cell membranes and/or a toxin principle within this layer may be responsible for fish mortality. Juvenile bay scallops (Argopecten irradians) and American oysters (Crassostrea virginica) experienced elevated mortality compared to control treatments during a 9-day exposure to bloom water (5 × 10⁴ cells ml⁻¹). Surviving scallops exposed to bloom water also experienced significantly reduced growth rates. Moribund shellfish displayed hyperplasia, hemorrhaging, squamation, and apoptosis in gill and digestive tissues with gill inflammation specifically associated with areas containing C. polykrikoides cells. In summary, our results indicate C. polykrikoides blooms have become annual events on eastern Long Island and that bloom waters are capable of causing rapid mortality in multiple species of finfish and shellfish.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Physiological acclimation to light in Chara intermedia nodes

In this study we investigated the ability of Chara intermedia to acclimate to different irradiances (i.e. “low-light” (LL): 20–30 μmol photons m⁻² s⁻¹ and “high-light” (HL): 180–200 μmol photons m⁻² s⁻¹) and light qualities (white, yellow and green), using morphological, photosynthesis, chlorophyll fluorescence and pigment analysis.Relative growth rates increased with increasing irradiance from 0.016 ± 0.003 (LL) to 0.024 ± 0.005 (HL) g g⁻¹ d⁻¹ fresh weight and were independent of light quality. A growth-based branch orientation towards high-light functioning as a mechanism to protect the plant from excessive light was confirmed. It was shown that the receptor responsible for the morphological reaction is sensitive to blue-light.C. intermedia showed higher oxygen evolution (up to 10.5 (HL) vs. 4.5 (LL) nmol O₂ mg Chl⁻¹ s⁻¹), photochemical and energy-dependent Chl fluorescence quenching and a lower Fv/Fm after acclimation to HL. With respect to qP, the acclimation of the photosynthetic apparatus depended on light quality and needed the blue part of the spectrum for full development. In addition, pigment composition was influenced by light and the Chl a/Car and Antheraxanthin (A) + Zeaxanthin (Z)/Violaxanthin (V) + A + Z (DES) ratios revealed the expected acclimation behaviour in favour of carotenoid protection under HL (i.e. decrease of Chl a/Car from 3.41 ± 0.48 to 2.30 ± 0.35 and increase of DES from 0.39 ± 0.05 to 0.87 ± 0.03), while the Chl a/Chl b ratios were not significantly affected. Furthermore it was shown that morphological light acclimation mechanisms influence the extent of the physiological modifications.     

Annual cycle of CO2 exchange over a reed (Phragmites australis) wetland in Northeast China

Net ecosystem exchange of CO₂ (NEE) was measured during 2005 using the eddy covariance (EC) technique over a reed (Phragmites australis (Cav.) Trin. ex Steud.) wetland in Northeast China (121°54′E, 41°08′N). Diurnal NEE patterns varied markedly among months. Outside the growing season, NEE lacked a diurnal pattern and it fluctuated above zero with an average value of 0.07 mg CO₂ m⁻² s⁻¹ resulting from soil microbial activity. During the growing season, NEE showed a distinct V-like diel course, and the mean daily NEE was −7.48 ± 2.74 g CO₂ m⁻² day⁻¹, ranging from −13.58 g CO₂ m⁻² day⁻¹ (July) to −0.10 g CO₂ m⁻² day⁻¹ (October). An annual cycle was also apparent, with CO₂ uptake increasing rapidly in May, peaking in July, and decreasing from August. Monthly cumulative NEE ranged from −115 ± 24 g C m⁻² month⁻¹ (the reed wetland was a CO₂ sink) in July to 75 ± 16 g C m⁻² month⁻¹ (CO₂ source) in November. The annual CO₂ balance suggests a net uptake of −65 ± 14 g C m⁻² year⁻¹, mainly due to the gains in June and July. Cumulative CO₂ emission during the non-growing season was 327 g C m⁻², much greater than the absolute value of the annual CO₂ balance, which proves the importance of the wintertime CO₂ efflux at the study site. The ratio of ecosystem respiration (R_eco) to gross primary productivity (GPP) for this reed ecosystem was 0.95, indicating that 95% of plant assimilation was consumed by the reed plant or supported the activities of heterotrophs in the soil. Daytime NEE values during the growing season were closely related to photosynthetically active radiation (PAR) (r² > 0.63, p < 0.01). Both maximum ecosystem photosynthesis rate (A_max) and apparent quantum yield (α) were season-dependent, and reached their peak values in July (1.28 ± 0.11 mg CO₂ m⁻² s⁻¹, 0.098 ± 0.027 μmol CO₂ μmol⁻¹ photon, respectively), corresponding to the observed maximum NEE in July. Ecosystem respiration (R_eco) relied on temperature and soil water content, and the mean value of Q₁₀ was about 2.4 with monthly variation ranging from 1.8 to 4.1 during 2005. Annual methane emission from this reed ecosystem was estimated to be about 3 g C m⁻² year⁻¹, and about 5% of the net carbon fixed by the reed wetland was released to the atmosphere as CH₄.     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of temperature, light intensity and photoperiod

Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d⁻¹ at 24 °C and 600 μmol m⁻² s⁻¹. Optimum growth rates (>0.4 d⁻¹) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m⁻² s⁻¹. The highest growth rate of C. fusus was 0.56 d⁻¹ at 26 °C and 216 μmol m⁻² s⁻¹. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m⁻² s⁻¹, and did not show photoinhibition at irradiances up to 796 μmol m⁻² s⁻¹. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.     

Phosphorus removal by the Ceratophyllum/periphyton complex in a south Florida (USA) freshwater marsh

Removal of phosphorus (P) by Ceratophyllum demersum L. and associated epiphytic periphyton was quantified by measuring the disappearance of soluble reactive P (SRP) from microcosms during 1-h in situ incubations conducted over a 1-year period. Initial P concentrations in these incubations ranged from 30 to >10,000 μg P L⁻¹. Phosphorus removal was proportional to initial P concentrations and was weakly correlated with solar irradiance and water temperature. Removal rates (0.6–32.8 mg P m⁻² d⁻¹) and k_v coefficients (0.68–1.93 h⁻¹) from experiments run at low initial P concentrations (up to 200 μg P L⁻¹) were comparable to results reported for other macrophytes. Removal rates from experiments run at the highest (>10,000 μg P L⁻¹) initial P concentrations (5300 and 11,100 mg P m⁻² d⁻¹) most likely represented luxury nutrient consumption and were not thought to be sustainable long term. We were unable to determine a V_max for P removal, suggesting that the nutrient-storage capability of the C. demersum/periphyton complex was not saturated during our short-term incubations. Based on N:P molar ratios, the marsh was P limited, while the C. demersum/periphyton complex was either N limited or in balance for N and P throughout this study. However, despite its tissue stoichiometry, the C. demersum/periphyton complex always exhibited an affinity for P. It appeared that the biochemical mechanisms, which mediate P removal, at least on a short-term basis, were more influenced by increases in ambient P levels than by tissue nutrient stoichiometry.     

The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup

The red tide dinoflagellate Karenia brevis (Davis) G. Hansen and Moestrup is noted for causing mass mortalities of marine organisms in the Gulf of Mexico. Most research has focused on culture isolates from the eastern Gulf of Mexico. In this investigation, we examine the effects of light, temperature and salinity on the growth rate of K. brevis from the western Gulf of Mexico. Growth rates of K. brevis were determined under various combinations of irradiance (19, 31, 52, 67, and 123 μmol m⁻² s⁻¹), salinity (25, 30, 35, 40 and 45), and temperature (15, 20, 25, and 30 °C). Maximum growth rates varied from 0.17 to 0.36 div day⁻¹ with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 19 μmol photons m⁻² s⁻¹ for any experiment. Maximum growth rates at 15 °C were much lower than at other temperatures. Maximum growth rates of the Texas clone (SP3) fell within the range of Florida clones reported in the literature (0.17–0.36 div day⁻¹ versus 0.2–1.0 div day⁻¹). The Texas clone SP3 had a very similar light saturation point compared to that of a Florida isolate (Wilson's clone) (67 μmol m⁻² s⁻¹ versus 65 μmol m⁻² s⁻¹), and light compensation (20–30 μmol m⁻² s⁻¹1). The upper and lower salinity tolerance of the Texas clone was similar than that of some Florida clones (45 versus 46 and 25 versus 22.5, respectively). In our study, the Texas clone had the same temperature tolerance reported for Florida clones (15–30 °C). While individual clones can vary considerably in maximum growth rates, our results indicate only minor differences exist between the Texas and Florida strains of K. brevis in their temperature and salinity tolerance for growth. While the literature notes lower salinity occurrences of K. brevis in nearby Louisiana, our isolate from the southern Texas coast has the higher salinity requirements typical of K. brevis in the eastern Gulf of Mexico.     

Characterization of the affinity for nitrogen, uptake kinetics, and environmental relationships for Prorocentrum minimum in natural blooms and laboratory cultures

During the late spring and early summer of 1998, an extensive bloom of the dinoflagellate Prorocentrum minimum (>93% of phytoplankton cell density) developed in several tributaries of the Chesapeake Bay, USA. In January 1999, a bloom of mixed dinoflagellates (Heterocapsa rotundata, H. triquetra and P. minimum, with P. minimum forming 21% of total phytoplankton cells and 39% of the total biovolume) developed in the mesohaline Neuse Estuary, North Carolina, USA. During these blooms, experiments were carried out to characterize the nitrogen uptake kinetics of these assemblages with ¹⁵N isotopic techniques. Four nitrogenous substrates (NO₃⁻, NH₄⁺, urea, and a mixed amino acids substrate) were used to determine uptake rate and substrate preference. Rates of nitrogen uptake were also measured in P. minimum cultures grown on varying growth nitrogen substrates. The calculated kinetic parameters determined for the P. minimum-dominated field assemblages and the cultures indicated a preference for NH₄⁺. NH₄⁺ was also the primary nitrogen source supporting the blooms. In addition, a high affinity for urea was also found, and urea contributed significantly to the Neuse Estuary bloom. Furthermore, results showed that the regulation of uptake for each of the substrates was different: strong positive relationships between affinity and temperature were found for NH₄⁺ and amino acids, while a negative response was found for NO₃⁻, and very little response to temperature was noted for urea. These differences suggest that a diversity of nitrogen uptake mechanisms may aid the development and maintenance of P. minimum blooms.     

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09 ± 0.02 μmol O₂ h^− 1 g^− 1; summer: 0.31 ± 0.06 μmol O₂ h^− 1 g^− 1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content / ascorbate content ratio (A / AH⁻). The A / AH⁻ ratio showed minimum values in winter (3.7 ± 0.2 10^− 5 AU) and increased in summer (18 ± 5 10^− 5 AU). A similar pattern was observed for lipid radical content (122 ± 29 pmol mg^− 1 fresh mass [FW] in winter and 314 ± 45 pmol mg^− 1 FW in summer), iron content (0.99 ± 0.07 and 2.7 ± 0.6 nmol mg^− 1 FW in winter and summer, respectively) and catalase activity (2.9 ± 0.2 and 7 ± 1 U mg^− 1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe–MGD–NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.