Growth-promoting effects of a bacterium on raphidophytes and other phytoplankton

On 29 April 2003, a Heterosigma akashiwo bloom (9.5 × 10⁴ cells mL⁻¹) associated with a fish kill (>10⁴ dead fishes estimated from aerial surveys) was observed offshore of Bulls Bay, McLellanville, South Carolina, USA. To assess a potential cause of this bloom event, we investigated the bacterial diversity and algal/bacterial interactions in the bloom microbial community. Thirty-five bacterial strains were isolated and screened for algicidal or algal growth-promoting activities. One strain (BBB25) had significant growth-promoting effects on all 7 algal species tested: three raphidophytes (Heterosigma akashiwo, Chattonella subsalsa, Fibrocapsa japonica), two diatoms (Chaetoceros neogracile, Nitzschia sp.), a cryptophyte (Cryptomonas sp.), and a chlorophyte, Ankistrodesmus sp. This strain (BBB25) is a Gram-positive, rod-shaped spore-forming bacterium. Partial 16S rDNA gene sequence and morphological characters indicated that BBB25 is related closely to the genus Bacillus. The general nature of the algal response indicates that the growth-promoting effects of BBB25 are not specific to H. akashiwo, and suggests potentially widespread effects. Since the presence or relative abundance of the other algal species was not assessed during the bloom initiation period, the selective stimulatory effect on H. akashiwo bloom formation in Bulls Bay is unknown. These results demonstrate, however, the potential for bacterial species to play a regulatory role in bloom formation.     

Toxigenic Pfiesteria species—Updates on biology,ecology, toxins,and impacts

The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 10² to 10³ (rarely, 10⁴) flagellate cells mL⁻¹. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (<10 cells mL⁻¹), and the available evidence suggests that the populations were mostly in the lower water column and within surficial sediments. Apparently highly sensitive to scouring effects from major storms, Pfiesteria populations have been sparse in the affected estuaries since several hurricanes struck the Albemarle-Pamlico in the late 1990s. Recent research highlights include characterization of a novel group of Pfiesteria toxins, culture of a toxigenic strain on a sterile fish cell line, axenic culture on a semi-defined medium, the discovery of a new mode of heterotrophic feeding in dinoflagellates as manifested by Pfiesteria, and other advances in understanding the nutritional ecology and prey acquisition of these harmful dinoflagellates.     

Algicidal and growth-inhibiting bacteria associated with seagrass and macroalgae beds in Puget Sound,WA, USA

The algicidal and growth-inhibiting bacteria associated with seagrasses and macroalgae were characterized during the summer of 2012 and 2013 throughout Puget Sound, WA, USA. In 2012, Heterosigma akashiwo-killing bacteria were observed in concentrations of 2.8 × 10⁶ CFU g⁻¹ wet in the outer organic layer (biofilm) on the common eelgrass (Zostera marina) in north Padilla Bay. Bacteria that inhibited the growth of Alexandrium tamarense were detected within the biofilm formed on the eelgrass canopy at Dumas Bay and North Bay at densities of ∼10⁸ CFU g⁻¹ wet weight. Additionally, up to 4100 CFU mL⁻¹ of algicidal and growth-inhibiting bacteria affecting both A. tamarense and H. akashiwo were detected in seawater adjacent to seven different eelgrass beds. In 2013, H. akashiwo-killing bacteria were found on Z. marina and Ulva lactuca with the highest densities of ∼10⁸ CFU g⁻¹ wet weight at Shallow Bay, Sucia Island. Bacteria that inhibited the growth of H. akashiwo and A. tamarense were also detected on Z. marina and Z. japonica at central Padilla Bay. Heterosigma akashiwo cysts were detected at a concentration of 3400 cysts g⁻¹ wet weight in the sediment from Westcott Bay (northern San Juan Island), a location where eelgrass disappeared in 2002. These findings provide new insights on the ecology of algicidal and growth-inhibiting bacteria, and suggest that seagrass and macroalgae provide an environment that may influence the abundance of harmful algae in this region. This work highlights the importance of protection and restoration of native seagrasses and macroalgae in nearshore environments, in particular those regions where shellfish restoration initiatives are in place to satisfy a growing demand for seafood.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Feeding by the newly described heterotrophic dinoflagellate Stoeckeria changwonensis: A comparison with other species in the family Pfiesteriaceae

The feeding ecology of the newly described heterotrophic dinoflagellate Stoeckeria changwonensis was explored. The feeding behavior of S. changwonensis, and the kinds of prey species that it feeds on were investigated with several different types of microscopes and high-resolution video-microscopy. Additionally, the growth and ingestion rates of S. changwonensis as a function of prey concentration for perch (Lateolabrax japonicus) blood cells, the raphidophyte Heterosigma akashiwo, the cryptophytes Rhodomonas salina and Teleaulax sp., and the phototrophic dinoflagellate Amphidinium carterae prey were measured. S. changwonensis feeds on prey through a peduncle, after anchoring the prey by using a tow filament. This type of feeding behavior is similar to that of Stoeckeria algicida, Pfiesteria piscicida, and Luciella masanensis in the family Pfiesteriaceae; however, S. changwonensis feeds on various kinds of prey species different from those of the other heterotrophic dinoflagellates. S. changwonensis ingested perch blood cells and diverse algal species, in particular, the large thecate dinoflagellate Lingulodinium polyedrum which are not eaten by the other peduncle feeders. H. akashiwo and the perch blood cells supported positive growth of S. changwonensis, but R. salina, Teleaulax sp., and A. carterae which support positive growth of P. piscicida and L. masanensis did not support positive growth of S. changwonensis. With increasing mean prey concentration the growth rates for S. changwonensis on H. akashiwo and the perch blood cells increased rapidly and then slowly or became saturated. The maximum growth rates of S. changwonensis on H. akashiwo and the perch blood cells were 0.376 and 0.354 d⁻¹, respectively. Further, the maximum ingestion rates of S. changwonensis on H. akashiwo and the perch blood cells were 0.35 ng C predator⁻¹ d⁻¹ (3.5 cells predator⁻¹ d⁻¹) and 0.27 ng C predator⁻¹ d⁻¹ (29 cells predator⁻¹ d⁻¹), respectively. These maximum growth and ingestion rates of S. changwonensis on H. akashiwo, the perch blood cells, R. salina, Teleaulax sp., and A. carterae differed considerably from those of S. algicida, P. piscicida, and L. masanensis on the same prey species. Thus, the feeding behavior of S. changwonensis may differ from that of other species in the family Pfiesteriaceae.     

pH alters the swimming behaviors of the raphidophyte Heterosigma akashiwo: Implications for bloom formation in an acidified ocean

We investigated the effects of pH on movement behaviors of the harmful algal bloom causing raphidophyte Heterosigma akashiwo. Motility parameters from >8000 swimming tracks of individual cells were quantified using 3D digital video analysis over a 6-h period in 3 pH treatments reflecting marine carbonate chemistry during the pre-industrial era, currently, and the year 2100. Movement behaviors were investigated in two different acclimation-to-target-pH conditions: instantaneous exposure and acclimation of cells for at least 11 generations. There was no negative impairment of cell motility when exposed to elevated PCO₂ (i.e., low pH) conditions but there were significant behavioral responses. Irrespective of acclimation condition, lower pH significantly increased downward velocity and frequency of downward swimming cells (p < 0.001). Rapid exposure to lower pH resulted in 9% faster downward vertical velocity and up to 19% more cells swimming downwards (p < 0.001). Compared to pH-shock experiments, pre-acclimation of cells to target pH resulted in ~30% faster swimming speed and up to 46% faster downward velocities (all p < 0.001). The effect of year 2100 PCO₂ levels on population diffusivity in pre-acclimated cultures was >2-fold greater than in pH-shock treatments (2.2 × 10⁵ μm² s⁻¹ vs. 8.4 × 10⁴ μm² s⁻¹). Predictions from an advection-diffusion model, suggest that as PCO₂ increased the fraction of the population aggregated at the surface declined, and moved deeper in the water column. Enhanced downward swimming of H. akashiwo at low pH suggests that these behavioral responses to elevated PCO₂ could reduce the likelihood of dense surface slick formation of H. akashiwo through reductions in light exposure or growth independent surface aggregations. We hypothesize that the HAB alga's response to higher PCO₂ may exploit the signaling function of high PCO₂ as indicative of net heterotrophy in the system, thus indicative of high predation rates or depletion of nutrients.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum

A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).     

Multiannual Chattonella subsalsa Biecheler (Raphidophyceae) blooms in a Mediterranean lagoon (Santa Giusta Lagoon,Sardinia Island,Italy)

Recurrent blooms of Chattonella subsalsa (Raphidophyceae) were associated with fish kills in Santa Giusta Lagoon (Mediterranean Sea). This study investigated the population dynamics of C. subsalsa and its relationship with environmental and meteorological conditions, using multiannual ecological data (1990–2016). In addition, for the first time, this study examined the presence of C. subsalsa cysts in lagoon sediments. The species was first detected in Santa Giusta Lagoon in July 1994. Bloom events coinciding with fish kills were recorded in 1994, 1998, 1999, and 2010. The timing and dynamics of C. subsalsa blooms and fish kills varied over the examined period. Presence of C. subsalsa was strongly influenced by temperature, especially in the early years of the series (1990–2002). Temperature control may have been lesser important in the more recent years, when higher temperature may have generated continuative suitable conditions for C. subsalsa affirmation, especially in July. Thus, the variations in the availability of food (via autotrophy and/or mixotrophy) could be one of the control keys on the proliferation of this species in the future in SG. Cysts of C. subsalsa were present in lagoon sediments at abundances ranging 200–2000 cysts g⁻¹ wet sediment. This study is among a few that have examined C. subsalsa population dynamics and bloom events in the field over a long time period. Findings from this study contribute to a better understanding of C. subsalsa bloom development, by identifying environmental and meteorological variables that may promote blooms of this species in the Santa Giusta Lagoon.     

Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes

The dinoflagellate community present during blooms of the fish killing dinoflagellate Cochlodinium polykrikoides was characterized by DNA melting curve analysis and direct sequencing of the SSU rDNA amplified from environmental sample extracts. PCR amplification of genomic DNA from Gaedo water samples using dinoflagellate-specific SSU rDNA primers yielded 280 clones, which were screened by closed tube PCR-melting curve analysis targeting a region of the SSU rDNA, enabling high throughput analysis. Twenty-eight clones producing distinct melting curve patterns were sequenced, and their phylogenetic information revealed that C. polykrikoides co-occurred with morphologically similar species including Gymnodinium impudicum and Gymnodinium catenatum. Temporal variations of C. polykrikoides and G. impudicum abundances in South Sea were also examined by species-specific real-time TaqMan-based PCR probes developed in this study. C. polykrikoides- and G. impudicum-specific real-time PCR probes were designed targeting the internal transcribed spacer 2 ribosomal DNA region. The probe specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR products from environmental samples. The real-time PCR assays showed that C. polykrikoides cell densities peaked in August at 16,928 cells mL^?1, while G. impudicum was present at low abundances (below 25 cells mL^?1). Our amplified rDNA melting curve protocol provides a facile method for the characterization of the dinoflagellate community, and the real-time PCR assay could be an alternative method for rapid and sensitive enumeration of harmful dinoflagellates in the marine environment.     

Monitoring,management, and mitigation of Karenia blooms in the eastern Gulf of Mexico

Annual blooms of the toxic dinoflagellate Karenia brevis in the eastern Gulf of Mexico represent one of the most predictable global harmful algal bloom (HAB) events, yet remain amongst the most difficult HABs to effectively monitor for human and environmental health. Monitoring of Karenia blooms is necessary for a variety of precautionary, management and predictive purposes. These include the protection of public health from exposure to aerosolized brevetoxins and the consumption of toxic shellfish, the protection and management of environmental resources, the prevention of bloom associated economic losses, and the evaluation of long term ecosystem trends and for potential future bloom forecasting and prediction purposes. The multipurpose nature of Karenia monitoring, the large areas over which blooms occur, the large range of Karenia cell concentrations (from 5 × 10³ cells L^?1 to >1 × 10⁶ cells L^?1) over which multiple bloom impacts are possible, and limitations in resources and knowledge of bloom ecology have complicated K. brevis monitoring, mitigation and management strategies. Historically, K. brevis blooms were informally and intermittently monitored on an event response basis in Florida, usually in the later bloom stages after impacts (e.g. fish kills, marine mammal mortalities, respiratory irritation) were noted and when resources were available. Monitoring of different K. brevis bloom stages remains the most practical method for predicting human health impacts and is currently accomplished by the state of Florida via direct microscopic counts of water samples from a state coordinated volunteer HAB monitoring program. K. brevis cell concentrations are mapped weekly and disseminated to stakeholders via e-mail, web and toll-free phone numbers and provided to Florida Department of Agriculture and Consumer Services (FDACS) for management of both recreational and commercial shellfish beds in Florida and to the National Oceanic and Atmospheric Administration (NOAA) for validation of the NOAA Gulf of Mexico HAB bulletin for provision to environmental managers. Many challenges remain for effective monitoring and management of Karenia blooms, however, including incorporating impact specific monitoring for the diverse array of potential human and environmental impacts associated with blooms, timely detection of offshore bloom initiation, sampling of the large geographic extent of blooms which often covers multiple state boundaries, and the involvement of multiple Karenia species other than K. brevis (several of which have yet to be isolated and described) with unknown toxin profiles. The implementation and integration of a diverse array of optical, molecular and hybrid Karenia detection technologies currently under development into appropriate regulatory and non-regulatory monitoring formats represents a further unique challenge.     

Characterization of the toxicity of Cochlodinium polykrikoides isolates from Northeast US estuaries to finfish and shellfish

Harmful algal blooms caused by Cochlodinium polykrikoides are annual occurrences in coastal systems around the world. In New York (NY), USA, estuaries, bloom densities range from 10³ to 10⁵ mL^?1 with higher densities (≥10⁴ cells mL^?1) being acutely toxic to multiple fish and shellfish species. Here, we report on the toxicity of C. polykrikoides strains recently isolated from New York and Massachusetts (USA) estuaries to juvenile fish (Cyprinodon variegates) and bay scallops (Argopecten irradians), as well as on potential mechanisms of toxicity. Cultures of C. polykrikoides exhibited dramatically more potent ichthyotoxicity than raw bloom water with 100% fish mortality occurring within ～1 h at densities as low as 3.3 × 10² cells mL^?1. More potent toxicity in culture was also observed in bioassays using juvenile bay scallops, which experienced 100% mortality during 3 days exposure to cultures at cell densities an order of magnitude lower than raw bloom water (～3 × 10³ cells mL^?1). The toxic activity per C. polykrikoides cell was dependent on the growth stages of cultures with early exponential growth cultures being more potent than cultures in late-exponential or stationary phases. The ichthyotoxicity of cultures was also dependent on both cell density and fish size, as a hyperbolic relationship between the death time of fish and the ratio of algal cell density to length of fish was found (～10³ cells mL^?1 cm^?1 yielded 100% fish mortality in 24 h). Simultaneous exposure of fish to C. polykrikoides and a second algal species (Rhodomonas salina or Prorocentrum minimum) increased survival time of fish, and decreased the fish mortality suggesting additional cellular biomass mitigated the ichthyotoxicity. Frozen and thawed-, sonicated-, or heat-killed-, C. polykrikoides cultures did not cause fish mortality. In contrast, cell-free culture medium connected to an active culture through a 5 μm nylon membrane caused complete mortality in fish, although the time required to kill fish was significantly longer than direct exposure to the whole culture. These results indicate that ichthyotoxicity of C. polykrikoides isolates is dependent on viability of cells and that direct physical contact between fish and cells is not required to cause mortality. The ability of the enzymes peroxidase and catalase to significantly reduce the toxicity of live cultures and the inability of hydrogen peroxide to mimic the ichthyotoxicity of C. polykrikoides isolates suggests that the toxicity could be caused by non-hydrogen peroxide, highly reactive, labile toxins such as ROS-like chemicals.     

Expansion of harmful brown tides caused by the pelagophyte,Aureoumbra lagunensis DeYoe et Stockwell,to the US east coast

Brown tides caused by the pelagophyte Aureoumbra lagunensis DeYoe et Stockwell have formed ecosystem disruptive algal blooms in shallow lagoons of Texas (TX), USA, for more than two decades but have never been reported elsewhere. During the summer of 2012, a dense brown tide occurred in the Mosquito Lagoon and northern Indian River Lagoon along the east coast of Florida (FL), USA. While chlorophyll a levels in this system have averaged 5 μg L⁻¹ during the past two decades, concentrations during this brown tide reached ∼200 μg L⁻¹. Concurrently, levels of nitrate were significantly lower than average and levels of dissolved organic nitrogen were significantly higher than average (p < 0.001 for both). Sequences of the 18S rRNA gene of the bloom community and of single cell isolates were identical to those of Aureoumbra lagunensis DeYoe et Stockwell from TX. The A. lagunensis brown tide in FL bloomed to densities exceeding 10⁶ cells mL⁻¹ (quantified with a species-specific immuno-label) from July through September, began to dissipate in October, but maintained densities exceeding 10⁵ cells mL⁻¹ in some regions through December of 2012. The decline of the bloom was associated with near-hypoxic conditions and more than 30 fish kills reported within the Mosquito Lagoon in September 2012, a number far exceeding all prior monthly reports in this system dating to 1996. Wild northern quahog populations (a.k.a. hard clam, Mercenaria mercenaria) suffered mass die offs during the brown tide and eastern oysters (Crassostrea virginica) that settled during 2012 were significantly smaller than prior years. Clearance rates of hard clams and eastern oyster were significantly reduced in the presence of Mosquito Lagoon bloom water and A. lagunensis monocultures isolated from the Mosquito Lagoon at densities of ∼10⁶ cells L⁻¹. The expansion of harmful brown tides caused by A. lagunensis to these estuaries represents a new threat to the US southeast coast.     

A novel Heterosigma akashiwo (Raphidophyceae) bloom extending from a South Carolina bay to offshore waters

In April 2003, a novel Heterosigma akashiwo bloom was observed that extended from Bulls Bay, South Carolina USA, to approximately 8 km offshore. The bloom was associated with a fish kill of approximately 10⁴ fish. The bloom coincided with salinities anomalously low for the region and optimal for H. akashiwo growth. The low salinities were related to the rediversion of freshwater a month earlier from the Cooper River into the Santee River, which partially feeds into Bulls Bay. H. akashiwo identification was confirmed using a species-specific real-time PCR assay modified for the direct amplification of target DNA from the bloom sample. Because this H. akashiwo bloom was associated with a fish kill, and exposure to bloom waters caused sublethal toxic effects on oysters, the resolution of the cause and potential recurrence of the bloom are of importance to fishery management.     

Role of selenium compounds on tyrosine hydroxylase activity,adrenomedullin and total RNA levels in hearts of rats

Synthetic organoselenium compounds can be tailored to achieve greater chemopreventive efficacy with minimal toxic side effects by structural modifications. Two organoselenium compounds (Se I and Se II) were synthesized and evaluated for their antihypertensive and therapeutic properties by adrenomedullin (ADM) levels and tyrosine hydroxylase (TH) activity assays in rat heart tissue. 7,12-Dimethylbenz[a]anthracene (DMBA) is known to generate DNA-reactive species during their metabolism, which may enhance oxidative stress in cells. TH is thought to be a rate-limiting enzyme in the biosynthesis of catecholamines. ADM, a potent endogenous vasodilating and natriuretic peptide, may play an important role in the pathophysiology of chronic heart failure. The effects of Se I and Se II were investigated on TH activity, ADM and total RNA levels in the hearts of albino Wistar rats. TH activity was found to be increased significantly by the effect of DMBA (P < 0.05). This increase was restricted in the Se I and Se II treated groups. ADM level was found to be decreased insignificantly by the effect of DMBA (P > 0.05). Total RNA level was found to be decreased significantly by the effect of DMBA (P < 0.05). This study demonstrates that synthetic organoselenium compounds can regulate DMBA-induced stress related changes in rat heart.     

Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation

Chemically stable nitroxide radicals that can be monitored by electron paramagnetic resonance (EPR) spectroscopy can provide information on structural and dynamic properties of functional RNA such as riboswitches. The convertible nucleoside approach is used to install 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl) labels at the exocyclic N⁴-amino group of cytidine and 2′-O-methylcytidine nucleotides in RNA. To obtain site-specifically labeled long riboswitch RNAs beyond the limit of solid-phase synthesis, we report the ligation of spin-labeled RNA using an in vitro selected deoxyribozyme as catalyst, and demonstrate the synthesis of TEMPO-labeled 53 nt SAM-III and 118 nt SAM-I riboswitch domains (SAM = S-adenosylmethionine).     

Ichthyotoxicity of four species of gymnodinioid dinoflagellates (Kareniaceae,Dinophyta) and purified karlotoxins to larval sheepshead minnow

Two species of Kareniaceae, Karlodinium veneficum (Swan and Huon River isolates) and Karlodinium conicum, and their respective purified karlotoxins (KmTx), were investigated for ichthyotoxicity on larval sheepshead minnow. Two non-karlotoxin producing species, Karenia mikimotoi and Karlodinium ballantinum were also tested. Algal treatments included live and lysed cells (homogenized and CuSO₄ treated) with fish mortalities observed from lysed Ka. veneficum and Ka. conicum but none observed from K. mikimotoi and Ka. ballantinum. The variance in ichthyotoxicity between live and lysed cells of Ka. veneficum (Swan and Huon River) and Ka. conicum (Southern Ocean) confirm that toxin is cell bound and ichthyotoxicity increases upon lysis. Ichthyotoxic blooms of Ka. veneficum in situ in the Swan River, Western Australia and Chesapeake Bay, Maryland, USA are unrelated to algal cell density as mortality was observed with low densities. In laboratory treatments, no fish mortalities were observed upon exposure to live intact cells of all four species at algal concentrations up to 2.5 × 10⁵ cells/mL in replete nutrient growth conditions. Lysed low density (3 × 10⁴ cells/mL) Ka. veneficum (Swan and Huon River) grown under P-limited nutrients caused quicker fish mortality than those cultured in replete nutrient conditions. Pure toxin isolated from Ka. veneficum (Swan and Huon River) and Ka. conicum (Southern Ocean) were toxic to sheepshead minnow larvae, with the lethal dose lowest for Km^HuonTx 2 (508.2 ng/mL), followed by Km^SwanTx 2-1 (563.2 ng/mL), and Km_conicumTx (762.4 ng/mL).