Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III under laboratory culture

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III, two species of causative bloom dinoflagellates in China, were investigated using bi-algal cultures under controlled laboratory conditions. The growth of P. donghaiense and S. trochoidea were significantly suppressed when the initial cell densities were set at 1.9 × 10⁴ cells mL⁻¹ or 1.9 × 10⁵ cells mL⁻¹ for P. donghaiense and 1.0 × 10⁴ cells mL⁻¹ for S. trochoidea when the initial size/density ratio was 1:1 or 10:1, respectively, but no out-competement was observed in either bi-algal culture by the end. The simultaneous assay on the culture filtrate showed that P. donghaiense filtrate prepared at a lower initial density (1.9 × 10⁴ cells mL⁻¹) stimulated the co-cultured S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹, but filtrate at a higher density (1.9 × 10⁵ cells mL⁻¹) depressed its growth. Differently, the filtrate of S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹ significantly suppressed the growth of P. donghaiense at a density of 1.9 × 10⁴ cells mL⁻¹, but had little stimulatory effect on P. donghaiense at a density of 1.9 × 10⁵ cells mL⁻¹compared to the control (P > 0.05). It is likely that these two species of microalgae interact with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. We then quantify their interactions in the bi-algal culture by using a mathematical model. The estimated parameters from the model showed that the inhibition exerted by S. trochoidea on P. donghaiense was about 43 and 24 times stronger than the inhibitory effect that P. donghaiense exerted on S. trochoidea when the initial size/density were 1:1 and 10:1, respectively. S. trochoidea seemed to have a survival strategy that was superior to P. donghaiense in the bi-algal culture under controlled laboratory conditions. We also observed a closely positive relationship between the initial cell density and its effect on the co-cultured microalga by measuring the fluorenscence: filtrate prepared from higher initial cell density had stronger interference on the co-cultured microalga. Moreover, pre-treated under different temperature conditions (30 °C, 60 °C and 100 °C) would significantly changed the effect of culture filtrate on the co-cultured microalga. Result inferred that P. donghaiense or S. trochoidea would release allelochemicals into the bi-algal culture medium and the allelochemicals might be a mixture with temperature-sensitive components in it.     

Occurrence and toxicity of Amphidinium carterae Hulburt in the North Arabian Sea

The occurrence and toxicity of Amphidinium carterae Hulburt is hereby reported for the first time from the North Arabian Sea on the coast of Pakistan. The concentrations of 1.2 × 10⁴ cells ml⁻¹ were found in intertidal pools that were also inhabited by the brown macroalga Sargassum wightii. Both wild and cultured A. carterae cells were tested for ciguatera toxicity through exposure to brine shrimp nauplii (Artemia salina) and albino mice. Although the brine shrimp did not appear to be affected mortalities in mice ranged between 13 and 16% at doses of 7.2 × 10⁴ and 2.5 × 10⁵ cells ml⁻¹, respectively. When mice were affected pharmacological effects such as muscle contraction in lower back area, increased respiration, immobility and paralysis in hind limbs were observed for 2 h. These effects appeared to be reversible and gradually disappeared within 24 h.     

Feeding by ciliates on two harmful algal bloom species, Prymnesium parvum and Prorocentrum minimum

We have focused on ciliates as potential grazers on toxic phytoplankton because they are major herbivores in aquatic food webs. Ciliates may exert top down control on toxic phytoplankton blooms, potentially suppressing or shortening the duration of harmful algal blooms (HABs). We measured the growth rates of several ciliate species on uni-algal and mixed diets of both HAB and non-HAB algae. The tintinnids Favella ehrenbergii, Eutintinnus pectinis and Metacylis angulata and the non-loricate ciliates Strombidinopsis sp. and Strombidium conicum were isolated from Long Island Sound (LIS), and fed HAB species including the prymnesiophyte Prymnesium parvum (strain 97-20-01) and the dinoflagellate Prorocentrum minimum (strains Exuv and JA 98-01). Ciliates were fed algal prey from cultures at various growth phases and at varying concentrations. We observed no harmful effects of P. minimum (Exuv) on any of the ciliates. However in a comparison of strains, P. minimum (Exuv) supported high growth rates, whereas P. minimum (JA 98-01) supported only nominal growth. P. parvum was acutely toxic to ciliates at high concentrations (2×10⁴–3×10⁴ cells ml⁻¹). At low concentrations (5×10³–1×10⁴ cells ml⁻¹), or in culture filtrate, ciliates survived for at least several hours. In mixed diet experiments, as long as a non-toxic alga was available, ciliates survived and at times grew well at concentrations of P. parvum (5×10²–3×10⁴ cells ml⁻¹) that would otherwise have killed them. The present study suggests that prior to the onset of toxicity and bloom formation ciliates may exert grazing pressure on these HAB species, potentially contributing to the suppression or decline of P. minimum and P. parvum blooms.     

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.     

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.     

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.     

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.     

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster^™ DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4±3.0×10⁶ and 5.4±3.0×10² CFU ml⁻¹ cell suspension. The detection limit was about 540 CFU ml⁻¹, which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4±3.0×10⁴ CFU g⁻¹ dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.     

Histopathology in Pacific oyster (Crassostrea gigas) spat caused by the dinoflagellate Prorocentrum rhathymum

The recognition of an apparent association between seasonal oyster spat mortalities (up to 40%) and high Prorocentrum rhathymum density in the Little Swanport Estuary, Tasmania, prompted further experimental investigation into the toxicity by this dinoflagellate. Standard brine shrimp, haemolysis assays and intraperitoneal mouse bioassays revealed fast acting toxins in methanol but not aqueous extracts of P. rhathymum, with mice dying in less than 20 min. Oyster bioassays involved feeding spat (4 mm shell width) for 21 consecutive days on a diet of cultured P. rhathymum at simulated bloom densities (10⁴ cells ml⁻¹). No oyster mortality was observed, however, histopathological signs of thin, dilated gut tubules and sloughing of gut cells resembled those seen in affected field samples. In contrast to field samples, gill pathology was also observed in experimental exposure oysters.     

Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species

A series of experiments was conducted to examine effects of four strains of the estuarine dinoflagellate, Pfiesteria shumwayae, on the behavior and survival of larval and adult shellfish (bay scallop, Argopecten irradians; eastern oyster, Crassostrea virginica; northern quahogs, Mercenaria mercenaria; green mussels, Perna viridis [adults only]). In separate trials with larvae of A. irradians, C. virginica, and M. mercenaria, an aggressive predatory response of three strains of algal- and fish-fed P. shumwayae was observed (exception, algal-fed strain 1024C). Larval mortality resulted primarily from damage inflicted by physical attack of the flagellated cells, and secondarily from Pfiesteria toxin, as demonstrated in larval C. virginica exposed to P. shumwayae with versus without direct physical contact. Survival of adult shellfish and grazing activity depended upon the species and the cell density, strain, and nutritional history of P. shumwayae. No mortality of the four shellfish species was noted after 24 h of exposure to algal- or fish-fed P. shumwayae (strains 1024C, 1048C, and CCMP2089) in separate trials at ≤5 × 10³ cells ml⁻¹, whereas higher densities of fish-fed, but not algal-fed, populations (>7–8 × 10³ cells ml⁻¹) induced low (≤15%) but significant mortality. Adults of all four shellfish species sustained >90% mortality when exposed to fish-fed strain 270A1 (8 × 10³ cells ml⁻¹). In contrast, adult M. mercenaria and P. viridis exposed to a similar density of fish-fed strain 2172C sustained <15% mortality, and there was no mortality of A. irradians and C. virginica exposed to that strain. In mouse bioassays with tissue homogenates (adductor muscle, mantle, and whole animals) of A. irradians and M. mercenaria that had been exposed to P. shumwayae (three strains, separate trials), mice experienced several minutes of disorientation followed by recovery. Mice injected with tissue extracts from control animals fed cryptomonads showed no response. Grazing rates of adult shellfish on P. shumwayae (mean cell length ±1 standard error [S.E.], 9 ± 1 μm) generally were significantly lower when fed fish-fed (toxic) populations than when fed populations that previously had been maintained on algal prey, and grazing rates were highest with the nontoxic cryptomonad, Storeatula major (cell length 7 ± 1 μm). Abundant cysts of P. shumwayae were found in fecal strands of all shellfish species tested, and ≤45% of the feces produced viable flagellated cells when placed into favorable culture conditions. These findings were supported by a field study wherein fecal strands collected from field-collected adult shellfish (C. virginica, M. mercenaria, and ribbed mussels, Geukensia demissa) were confirmed to contain cysts of P. shumwayae, and these cysts produced fish-killing flagellated populations in standardized fish bioassays. Thus, predatory feeding by flagellated cells of P. shumwayae can adversely affect survival of larval bivalve molluscs, and grazing can be depressed when adult shellfish are fed P. shumwayae. The data suggest that P. shumwayae could affect recruitment of larval shellfish in estuaries and aquaculture facilities; shellfish can be adversely affected via reduced filtration rates; and adult shellfish may be vectors of toxic P. shumwayae when shellfish are transported from one geographic location to another.     

Selective Control of the Prorocentrum minimum Harmful Algal Blooms by a Novel Algal-Lytic Bacterium Pseudoalteromonas haloplanktis AFMB-008041

In this study, we examined the algal-lytic activities and biological control mechanisms of Pseudoalteromonas haloplanktis AFMB-08041, which was isolated from surface seawater obtained at Masan Bay in Korea. In addition, we assessed whether AFMB-08041 could be used as a biocontrol agent to regulate harmful dinoflagellate Prorocentrum minimum. From these experiments, we found that the inoculation of AFMB-08041 at a final density of 2.5 × 10⁴ cfu ml⁻¹ caused P. minimum cells to degrade (>90%) within 5 days. The algal cells were lysed through an indirect attack by the AFMB-08041 bacterial strain. Our results also suggest that the algal-lytic compounds produced by AFMB-08041 may have β-glucosidase activity. However, P. haloplanktis AFMB-08041 was not able to suppress the growth of other alga such as Alexandrium tamarense, Akashiwo sanguinea, Cochlodinium polykrikoides, Gymnodinium catenatum, and Heterosigma akashiwo. Moreover, we observed that the growth of Prorocentrum dentatum, which has a very similar morphological structure to P. minimum, was also effectively suppressed by P. haloplanktis AFMB-08041. Therefore, the effect of AFMB-08041 on P. minimum degradation appears to be species specific. When testing in an indoor mesocosms, P. haloplanktis AFMB-08041 reduced the amount of viable P. minimum cells by 94.5% within 5 days after inoculation. The combined results of this study clearly demonstrate that this bacterium is capable of regulating the harmful algal blooms of P. minimum. In addition, these results will enable us to develop a new strategy for the anthropogenic control of harmful algal bloom-forming species in nature.     

Characteristics and seasonal variation in the semen of Markhoz bucks in western Iran

The aims of the present study were to evaluate if seasonality in semen characteristics and plasma testosterone concentrations exist in Markhoz male goats. Ten Markhoz (Angora) bucks were housed and fed according to standard recognized practices. During the observation period, semen was collected monthly with the aid of an electro-ejaculator and examined microscopically immediately after collection. Physical parameters of semen and the semen index were recorded. Blood samples were also taken monthly throughout the observation period and the plasma testosterone concentration monitored. Bucks demonstrated a higher semen quality (P < 0.05) in autumn and summer (semen index of 965 × 10⁶ and 752 × 10⁶ ml⁻¹, respectively), compared to spring and winter (semen index of 606 × 10⁶ and 512 × 10⁶, respectively). This coincided with a higher (P < 0.05) plasma testosterone concentration in autumn and summer (8.1 and 10.1 ng ml⁻¹, respectively), compared to that obtained in spring (3.0 ng ml⁻¹) and winter (2.5 ng ml⁻¹). During autumn and summer, the ejaculate volume (average of 1.2 and 1.0 ml), sperm output (1159 × 10⁶ and 1005 × 10⁶ sperm ml⁻¹), sperm mass motility (4.2 and 4.3), sperm progressive motility (83.9 and 82.0%) and percentage live sperm (90.7 and 88.2%, respectively) of the bucks were higher (P < 0.05) than in the spring (0.6 ml, 880 × 10⁶ sperm ml⁻¹, 3.3, 71.5% and 80.2%) and winter (0.7 ml, 863 × 10⁶ sperm ml⁻¹, 4.0, 71.5% and 84.9%, respectively). During autumn and summer, the percentage of sperm abnormalities (5.0 and 9.2%) was significantly lower than that in spring (12.9%) and winter (11.2%). The semen pH was slightly alkaline being significantly (P < 0.05) lower in the autumn (7.1) than in spring (7.3). Data showed season of the year to influence all semen parameters evaluated—indicating that optimal buck performance may be obtained in late summer and autumn. It can thus be said that Markhoz bucks have distinct seasonal spermatogenic activity, with poorer semen characteristics being recorded during winter and spring. This may be a critical obstacle when implementing an intensive breeding system of three kidding seasons in 2 years, with natural mating being implemented.     

Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape

A strain of Bacillus subtilis (NJ-18) with broad antimicrobial activity was screened in the laboratory and in the field. NJ-18 inhibited the in vitro radial extension of hyphae of the phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. The bacterium apparently produced antifungal metabolites that diffused through the agar and caused abnormal swelling of hyphae. The in vitro data and observations indicated that one of the mechanisms of inhibition by NJ-18 is antibiosis. In field experiments for control of sheath blight of rice, fermentation of NJ-18 at 5.0 × 10⁷ cfu ml⁻¹ significantly reduced disease incidence and severity; NJ-18 alone or combined with 50% kresoxim-methyl treatment at 225 g ai ha⁻¹ provided better control than 50% kresoxim-methyl at 225 g ai ha⁻¹ or Jinggangmycin at 120 g ai ha⁻¹, and control by NJ-18 alone was as high as 100.0%. In field experiments for control of Sclerotinia stem rot of rape, fermentation of NJ-18 at 1.0 × 10⁷ cfu ml⁻¹ again significantly reduced disease incidence and severity; control by NJ-18 was as high as 77.1% and was comparable with control by 46% dimethachlon and better than control by 50% carbendazim at 750 g ai ha⁻¹. We conclude that strain NJ-18 of B. subtilis is a promising biological control agent and should be further studied and tested for control of sheath blight of rice, Sclerotinia stem rot of rape, and other diseases.     

Ostracodology in time and space: looking back on fifteen International Symposia on Ostracoda,and the times in between

Nuclease protection assay (NPA) probes were designed to target the rRNA of Chaetoceros curvisetus and Skeletonema costatum, and quantitative sandwich hybridization integrated with nuclease protection assay (NPA-SH) was developed to detect C. curvisetus and S. costatum in culture and field samples in Jiaozhou Bay, China. The specificity and validity of the NPA-SH technique were tested with cultured pure strains, mixed strains and field samples, and by comparison with that of microscopy observation. The linear detection range for C. curvisetus was 4.2 × 10⁴ to 1.2 × 10⁶ cells with a detection limit of 42 cells ml⁻¹. The linear range for S. costatum was 6.0 × 10⁴ to 1.0 × 10⁶ cells with a detection limit of 60 cells ml⁻¹. The NPA-SH in this study provides a convenient tool for rapid assessment of HAB species in marine environments. Handling editor: D. Hamilton     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Use of a real-time remote monitoring network (RTRM) and shipborne sampling to characterize a dinoflagellate bloom in the Neuse Estuary, North Carolina, USA

The spatial-temporal distribution of a dinoflagellate bloom dominated or co-dominated by Prorocentrum minimum was examined during autumn through early spring in a warm temperate, eutrophic estuary. The developing bloom was first detected from a web-based alert provided by a network of real-time remote monitoring (RTRM) platforms indicating elevated dissolved oxygen and pH levels in upper reaches of the estuary. RTRM data were used to augment shipboard sampling, allowing for an in-depth characterization of bloom initiation, development, movement, and dissipation. Prolonged drought conditions leading to elevated salinities, and relatively high nutrient concentrations from upstream inputs and other sources, likely pre-disposed the upper estuary for bloom development. Over a 7-month period (October 2001–April 2002), the bloom moved toward the northern shore of the mesohaline estuary, intensified under favorable conditions, and finally dissipated after a major storm. Bloom location and transport were influenced by prevailing wind structure and periods of elevated rainfall. Chlorophyll a within bloom areas averaged 106 ± 13 μg L⁻¹ (mean ± 1 S.E.; maximum, 803 μg L⁻¹), in comparison to 20 ± 1 μg L⁻¹ outside the bloom. There were significant positive relationships between dinoflagellate abundance and TN and TP. Ammonium, NO₃⁻, and SRP concentrations did not decrease within the main bloom, suggesting that upstream inputs and other sources provided nutrient-replete conditions. In addition, PAM fluorometric measurements (09:00–13:00 h) of maximal PSII quantum yield (F_v/F_m) were consistently 0.6–0.8 within the bloom until late March, providing little evidence of photo-physiological stress as would have been expected under nutrient-limiting conditions. Nitrogen uptake kinetics were estimated for P. minimum during the period when that species was dominant (October–December 2001), based on literature values for N uptake by an earlier P. minimum bloom (winter 1999) in the Neuse Estuary. The analysis suggests that NH₄⁺ was the major N species that supported the bloom. Considering the chlorophyll a concentrations during October and December and the estimated N uptake rates, phytoplankton biomass was estimated to have doubled once per day. Bloom displacement (January–February) coincided with higher diversity of heterotrophic dinoflagellate species as P. minimum abundance decreased. This research shows the value of RTRM in bloom detection and tracking, and advances understanding of dinoflagellate bloom dynamics in eutrophic estuaries.     

Phytoplankton and bacterial assemblages in ballast water of U.S. military ships as a function of port of origin, voyage time, and ocean exchange practices

We characterized the physical/chemical conditions and the algal and bacterial assemblages in ballast water from 62 ballast tanks aboard 28 ships operated by the U.S. Military Sealift Command and the Maritime Administration, sampled at 9 ports on the U.S. West Coast and 4 ports on the U.S. East Coast. The ballast tank waters had been held for 2–176 days, and 90% of the tanks had undergone ballast exchange with open ocean waters. Phytoplankton abundance was highly variable (grand mean for all tanks, 3.21 × 10⁴ viable cells m⁻³; median, 7.9 × 10³ cells m⁻³) and was unrelated to physical/chemical parameters, except for a positive relationship between centric diatom abundance and nitrate concentration. A total of 100 phytoplankton species were identified from the ballast tanks, including 23 potentially harmful taxa (e.g. Chaetoceros concavicornis, Dinophysis acuminata, Gambierdiscus toxicus, Heterosigma akashiwo, Karlodinium veneficum, Prorocentrum minimum, Pseudo-nitzschia multiseries). Assemblages were dominated by chain-forming diatoms and dinoflagellates, and viable organisms comprised about half of the total cells. Species richness was higher in ballast tanks with coastal water, and in tanks containing Atlantic or Pacific Ocean source waters rather than Indian Ocean water. Total and viable phytoplankton numbers decreased with age of water in the tanks. Diversity also generally decreased with water age, and tanks with ballast water age >33 days did not produce culturable phytoplankton. Abundance was significantly higher in tanks with recently added coastal water than in tanks without coastal sources, but highly variable in waters held less than 30 days. Bacterial abundance was significantly lower in ballast tanks with Atlantic than Pacific Ocean source water, but otherwise was surprisingly consistent among ballast tanks (overall mean across all tanks, 3.13 ± 1.27 × 10¹¹ cells m⁻³; median, 2.79 × 10¹¹ cells m⁻³) and was unrelated to vessel type, exchange status, age of water, environmental conditions measured, or phytoplankton abundance. At least one of four pathogenic eubacteria (Listeria monocytogenes, Escherichia coli, Mycobacterium spp., Pseudomonas aeruginosa) was detected in 48% of the ballast tanks, but toxigenic strains of Vibrio cholerae were not detected. For ships with tanks of similar ballasting history, the largest source of variation in phytoplankton and bacteria abundance was among ships; for ships with tanks of differing ballasting histories, and for all ships/tanks considered collectively, the largest source of variation was within ships. Significant differences in phytoplankton abundance, but not bacterial abundance, sometimes occurred between paired tanks with similar ballasting history; hence, for regulatory purposes phytoplankton abundance cannot be estimated from single tanks only. Most tanks (94%) had adequate records to determine the source locations and age of the ballast water and, as mentioned, 90% had had ballast exchange with open-ocean waters. Although additional data are needed from sediments that can accumulate at the bottom of ballast tanks, the data from this water-column study indicate that in general, U.S. Department of Defense (DoD) ships are well managed to minimize the risk for introduction of harmful microbiota. Nevertheless, abundances of viable phytoplankton with maximum dimension >50 μm exceeded proposed International Maritime Organization standards in 47% of the ballast tanks sampled. The data suggest that further treatment technologies and/or alternative management strategies will be necessary to enable DoD vessels to comply with proposed standards.     

The development and multiple uses of a standardised bioassay method to select hypocrealean fungi for biological control of aphids

A technically standardised bioassay method was designed, evaluated and used to assess virulence and host range of hypocrealean fungi against aphids. A track mounted sprayer was used to apply conidia because hand held versions of the same sprayer can be used for field applications, thereby allowing the outcome from laboratory experiments to predict activity in the field accurately. Eighteen fungal isolates were assessed in single concentration bioassays against the black bean aphid Aphis fabae Scopoli. Isolates comprised commercially available mycoinsecticides (based on Beauveria bassiana and Lecanicillium longisporum) and isolates of B. bassiana, Lecanicillium spp., Paecilomyces fumosoroseus and Metarhizium anisopliae. Aphid mortality was in excess of 80% for 15 isolates, and HRI 1.72 (L. longipsorum), Z11 (P. fumosoroseus), Mycotech strain GHA (B. bassiana) and ARSEF 2879 (B. bassiana) were studied further. Multiple concentration bioassays identified HRI 1.72 as the most virulent isolate against A. fabae with significantly smaller LC₅₀ and LT₅₀ values compared to other isolates. A precise LC₅₀ value (2.95 × 10² conidia ml⁻¹) was calculated for HRI 1.72 using a second multiple concentration assay with smaller concentrations of conidia. The four isolates were applied at a single concentration (1 × 10⁸ conidia ml⁻¹) against Myzus persicae, A. fabae, Acyrthosiphon pisum, Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi. A ranking of aphid susceptibility was obtained, such that S. avenae > M. persicae, A. pisum, A. fabae > R. padi. Results indicate the importance of standardising bioassay methods to reduce bioassay variability without compromising the ability to use the bioassay to investigate fungus–host interactions under varying abiotic and biotic conditions.     

Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists

The marine dinoflagellate genus Alexandrium (Halim) Balech contains members that produce highly potent phycotoxins (PSP toxins or spirolides) as well as lytic substances and other allelochemicals of unknown structure and ecological significance. One isolate each of six Alexandrium species (A. tamarense, A. ostenfeldii, A. lusitanicum, A. minutum, A. catenella, A. taylori), of the closely related gonyaulacoid dinoflagellate Fragilidium subglobosum, and of the peridinioid Scrippsiella trochoidea were tested in 24 h co-incubation experiments for their short-term deleterious effects on a diversity of marine protists. Both autotrophs (Rhodomonas salina, Dunaliella salina, Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina, Amphidinium crassum, Rimostrombidium caudatum) were included as target species. All donor isolates except S. trochoidea exhibited lytic effects on at least some target species. Lytic effects were observed with all Alexandrium species, for both whole cell samples and culture filtrate (<10 μm and <0.2 μm). Antibiotic treated cultures with drastically reduced bacterial numbers did not show any general reduction in lytic capacity, therefore direct involvement of extracellular bacteria in allelochemical production is unlikely. Values of EC₅₀, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, differed by two orders of magnitude depending on the donor/target combination, from 3.1 × 10³ cells ml⁻¹ (A. minutum/O. marina) down to 0.02 × 10³ cells ml⁻¹ (A. catenella/D. salina). Within the array of nine donor Alexandrium/target combinations, variable ratios in EC₅₀ values between donor/target combination cannot be explained by quantitative differences in allelochemical production, but rather indicate qualitative differences in the composition of compounds produced by different Alexandrium strains. In conclusion, our study confirms the widespread lytic capacity within the genus Alexandrium, although allelochemical effects are not restricted to this genus. Allelochemical interactions mediated by such lytic substances may be significant in explaining the formation and maintenance of Alexandrium blooms through direct destructive effects on competing algae or unicellular grazers.