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.     

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.     

Killing potential protist predators as a survival strategy of the newly described dinoflagellate Alexandrium pohangense

Blooms caused by some species belonging to the dinoflagellate genus Alexandrium are known to cause large-scale mortality of fish. Thus, the dynamics of these species is important and of concern to scientists, officials, and people in the aquaculture industry. To understand the dynamics of such species, their growth and mortality due to predation need to be assessed. The newly described dinoflagellate Alexandrium pohangense is known to grow slowly, with a maximum autotrophic growth rate of 0.1 d⁻¹. Thus, it may not form bloom patches if its mortality due to predation is high. Therefore, to explore the mortality of A. pohangense due to predation, feeding on this species by the common heterotrophic dinoflagellates Gyrodinium dominans, Gyrodinium moestrupii, Luciella masanensis, Noctiluca scintillans, Oxyrrhis marina, Oblea rotunda, Polykrikos kofoidii, and Pfiesteria piscicida, as well as by the ciliate Tiarina fusus, was examined. None of these potential predators was able to feed on A. pohangense. In contrast, these potential predators were killed and their bodies were dissolved when incubated with A. pohangense cells or cell-free culture filtrates. The survival of G. moestrupii, O. marina, P. kofoidii, and T. fusus on incubation with 10 cells ml⁻¹ of A. pohangense was 20–60%, while that at the equivalent culture filtrates was 20–70%. With increasing A. pohangense cell-concentration (up to 1000 cells ml⁻¹ or equivalent culture filtrates), the survival rate of G. moestrupii, O. marina, P. kofoidii, and T. fusus rapidly decreased. The lethal concentration (LC₅₀) for G. moestrupii, O. marina, P. kofoidii, and T. fusus at the elapsed time of 24 h with A. pohangense cells (cultures of 11.4, 13.3, 1.6, and 3.3 cells ml⁻¹, respectively) was lower than that with A. pohangense filtrates (culture filtrates of 35.5, 30.6, 5.5, and 5.0 cells ml⁻¹, respectively). Furthermore, most of the ciliates and heterotrophic dinoflagellates in the water collected from the coast of Tongyoung, Korea, were killed when incubated with cultures of 1000 A. pohangense cells ml⁻¹ and equivalent culture filtrates. The relatively slow growing A. pohangense may form blooms by reducing mortality due to predation through killing potential protist predators.     

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.     

Differential interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and common heterotrophic protists and copepods: Killer or prey

To investigate interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and different heterotrophic protist and copepod species, feeding by common heterotrophic dinoflagellates (Oxyrrhis marina and Gyrodinium dominans), naked ciliates (Strobilidium sp. approximately 35 μm in cell length and Strombidinopsis sp. approximately 100 μm in cell length), and calanoid copepods Acartia spp. (A. hongi and A. omorii) on P. shiwhaense was explored. In addition, the feeding activities of P. shiwhaense on these heterotrophic protists were investigated. Furthermore, the growth and ingestion rates of O. marina, G. dominans, Strobilidium sp., Strombidinopsis sp., and Acartia spp. as a function of P. shiwhaense concentration were measured. O. marina, G. dominans, and Strombidinopsis sp. were able to feed on P. shiwhaense, but Strobilidium sp. was not. However, the growth rates of O. marina, G. dominans, Strobilidium sp., and Strombidinopsis sp. feeding on P. shiwhaense were very low or negative at almost all concentrations of P. shiwhaense. P. shiwhaense frequently fed on O. marina and Strobilidium sp., but did not feed on Strombidinopsis sp. and G. dominans. G. dominans cells swelled and became dead when incubated with filtrate from the experimental bottles (G. dominans + P. shiwhaense) that had been incubated for one day. The ingestion rates of O. marina, G. dominans, and Strobilidium sp. on P. shiwhaense were almost zero at all P. shiwhaense concentrations, while those of Strombidinopsis sp. increased with prey concentration. The maximum ingestion rate of Strombidinopsis sp. on P. shiwhaense was 5.3 ng C predator⁻¹d⁻¹ (41 cells predator⁻¹d⁻¹), which was much lower than ingestion rates reported in the literature for other mixotrophic dinoflagellate prey species. With increasing prey concentrations, the ingestion rates of Acartia spp. on P. shiwhaense increased up to 930 ng C ml⁻¹ (7180 cells ml⁻¹) at the highest prey concentration. The highest ingestion rate of Acartia spp. on P. shiwhaense was 4240 ng C predator⁻¹d⁻¹ (32,610 cells predator⁻¹d⁻¹), which is comparable to ingestion rates from previous studies on other dinoflagellate prey species calculated at similar prey concentrations. Thus, P. shiwhaense might play diverse ecological roles in marine planktonic communities by having an advantage over competing phytoplankton in anti-predation against potential protistan grazers.     

Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and insect larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28 ± 0.15) × 10⁶ U ml⁻¹ and (1.3 ± 0.2) × 10⁶ U ml⁻¹ respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7 ± 0.2) × 10⁶ U ml⁻¹ and (3.5 ± 0.4) × 10⁶ U ml⁻¹ at 2 dpi in Rachiplusia nu larvae and (1.1 ± 0.2) × 10⁶ U ml⁻¹ and (1.0 ± 0.15) × 10⁶ U ml⁻¹ at 5 dpi in Spodoptera frugiperda larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.     

Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

A mutualistic interaction between the bacterium Pseudomonas asplenii and the harmful algal species Chattonella marina (Raphidophyceae)

Several studies on various Chattonella species have reported that bacteria may play an important role in Chattonella bloom initiation, however, no studies have described how these bacteria promote the growth of C. marina. The interaction between C. marina and bacteria was investigated for identification and characterization of potential growth-promoting bacteria. In preliminary tests, the growth promoting effect of Pseudomonas species (25 strains) was investigated and P. asplenii (≥2.27) was determined as a growth-promoting bacteria for both C. marina strains (CCMP 2049 and 2050). This bacterium exerted optimal growth-promoting effects on C. marina, causing an increase in the initial density of P. asplenii to approximately 1 × 10⁷ cells mL⁻¹, which was used as the initial density in this study. To determine whether the growth-promoting activity was direct or indirect, P. asplenii was incubated in the algal media and then a filtrate of this culture was added to both C. marina strains. The P. asplenii filtrate stimulated the growth of C. marina and maintained the growth-promoting effects after high temperature (121 °C for 20 min) and pressure (15 psi) treatment. Thus, P. asplenii is able to promote C. marina growth through the release of a heat-resistant substance, such as inorganic nutrients. A nutrient analysis indicated that this bacterium elevated the phosphate concentration. Interestingly, P. asplenii was unable to survive in phosphate-limited media but could grow in phosphate-limited media incubating C. marina. Moreover, this bacterium could secrete significantly more phosphate in the presence of C. marina (p < 0.0001). These results suggested that P. asplenii and C. marina may have a mutualistic interaction.     

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 10⁶ cells ml⁻¹). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm⁻¹ could be measured without signal saturation of the Fogale Biomass system^®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC⁻¹ (0.15 × 10⁶ cells cm⁻²). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.     

Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L⁻¹ to 3.16 g L⁻¹, with a molecular weight range of 1.40×10⁶–1.83×10⁶ Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10⁶ U mL⁻¹), the production of HA was substantially increased from 5.96 g L⁻¹ to 19.38 g L⁻¹. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10³–1.42×10⁶ Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.     

Mixotrophy in the newly described dinoflagellate Yihiella yeosuensis: A small,fast dinoflagellate predator that grows mixotrophically,but not autotrophically

To investigate tropical roles of the newly described Yihiella yeosuensis (ca. 8 μm in cell size), one of the smallest phototrophic dinoflagellates in marine ecosystems, its trophic mode and the types of prey species that Y. yeosuensis can feed upon were explored. Growth and ingestion rates of Y. yeosuensis on its optimal prey, Pyramimonas sp. (Prasinophyceae), as a function of prey concentration were measured. Additionally, growth and ingestion rates of Y. yeosuensis on the other edible prey, Teleaulax sp. (Cryptophyceae), were also determined for a single prey concentration at which both these rates of Y. yeosuensis on Pyramimonas sp. were saturated. Among bacteria and diverse algal prey tested, Y. yeosuensis fed only on small Pyramimonas sp. and Teleaulax sp. (both cell sizes = 5.6 μm). With increasing mean prey concentrations, both specific growth and ingestion rates of Y. yeosuensis increased rapidly before saturating at a mean Pyramimonas concentration of 109 ng C mL⁻¹ (2725 cells mL⁻¹). The maximum growth rate (mixotrophic growth) of Y. yeosuensis fed with Pyramimonas sp. at 20 °C under a 14:10-h light-dark cycle of 20 μE m⁻² s⁻¹ was 1.32 d⁻¹, whereas the growth rate of Y. yeosuensis without added prey was 0.026 d⁻¹. The maximum ingestion rate of Y. yeosuensis fed with Pyramimonas sp. was 0.37 ng C predator⁻¹ d⁻¹ (9.3 cells predator⁻¹ d⁻¹). At a Teleaulax concentration of 1130 ng C mL⁻¹ (66,240 cells mL⁻¹), growth and ingestion rates of Y. yeosuensis fed with Teleaulax sp. were 1.285 d⁻¹ and 0.38 ng C predator⁻¹ d⁻¹ (22.4 cells predator⁻¹ d⁻¹), respectively. Thus, Y. yeosuensis rarely grows without mixotrophy, and mixotrophy supports high growth rates in Y. yeosuensis. Y. yeosuensis has the highest maximum mixotrophic growth rate with the exception of Ansanella graniferaamong engulfment feeding mixotrophic dinoflagellates. However, the high swimming speed of Y. yeosuensis (1572 μm s⁻¹), almost the highest among phototrophic dinoflagellates, may prevent autotrophic growth. This evidence suggests that Y. yeosuensis may be an effective mixotrophic dinoflagellate predator on Pyramimonas and Teleaulax, and occurs abundantly during or after blooms of these two prey species.     

Glucocorticoid receptors on and in a unicellular organism,Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml⁻¹ of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml⁻¹ than in cultures with 14,000 parasites ml⁻¹. Also, 100 ng ml⁻¹ of RU486/ml was more effective than 50 ng ml⁻¹ in inhibiting parasite multiplication in the 14,000 parasites ml^-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and the relationship with bloom occurrences in Kesennuma Bay,Japan

While cyst germination may be an important factor for the initiation of harmful/toxic blooms, assessments of the fluctuation in phytoplankton cyst germination, from bottom sediments to water columns, are rare in situ due to lack of technology that can detect germinated cells in natural bottom sediments. This study introduces a simple mesocosm method, modeled after previous in situ methods, to measure the germination of plankton resting stage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellates resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A. pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was conducted in addition to the typical samplings of seawater and bottom sediments to detect the dinoflagellates vegetative cells and resting cysts. Bloom occurrences of A. fundyense were observed June 2014 and February 2015 with maximum cell densities reaching 3.6 × 10⁶ cells m⁻² and 1.4 × 10⁷ cells m⁻², respectively. The maximum germination fluxes of A. fundyense cysts occurred in April 2014 and December 2014 and were 9.3 × 10³ cells m⁻² day⁻¹ and 1.4 × 10⁴ cells m⁻² day⁻¹, respectively. For A. pacificum, the highest cell density was 7.3 × 10⁷ cells m⁻² during the month of August, and the maximum germination fluxes occurred in July and August, reaching 5.8 × 10² cells m⁻² day⁻¹. Thus, this study revealed the seasonal dynamics of A. fundyense and A. pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred one to two months after peak germination, which strongly suggests that both the formation of the initial population by cyst germination and its continuous growth in the water column most likely contributed to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.     

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.     

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS–mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from ∼ 3–50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of ∼ 1.6 × 10⁷ CFU ml^− 1 (∼ 1.6 × 10⁴ cells spot^− 1), and bound to proteins of ∼ 50 and ∼ 39 kDa. Other MAbs, binding to proteins ranging from ∼ 3–14 and ∼ 40 kDa, detected VVB (but not VVC) with high sensitivity at ∼ 1.6 × 10⁵ and 4 × 10⁶ CFU ml^− 1 (∼ 1.6 × 10² and 4 × 10³ cells spot^− 1), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.     

Effect of Pichia membranaefaciens in combination with salicylic acid on postharvest blue and green mold decay in citrus fruits

The separate or combined effects of Pichia membranaefaciens and salicylic acid (SA) on the control of blue and green mold decay in citrus fruits were investigated. Results indicate that combining P. membranaefaciens (1 × 10⁸ CFU ml⁻¹) with SA (10 μg ml⁻¹) either in a point-inoculated or dipped treatment provided a more effective control of blue and green mold than separately applying yeast or SA. SA (10 μg ml⁻¹) did not significantly affect P. membranaefaciens growth in vitro but slightly increased the yeast population in fruit wounds. P. membranaefaciens plus SA effectively enhanced the phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, chitinase, and β-1,3-glucanase activities and stimulated the synthesis of phenolic compounds. The combined treatment did not impair quality parameters such as weight loss or titratable acidity, but resulted in low average natural infection incidence and increased total soluble solids and ascorbic acid contents in citrus fruits after 14 d at 20 °C.     

Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method

Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml⁻¹ eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml⁻¹ within 24 h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml⁻¹ to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24 h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800 cells ml⁻¹ A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48 h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1 m³ stock culture of A. pohangense at 4000 cells ml⁻¹ is calculated to remove all C. polykrikoides cells in ca. 200 m³ within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml⁻¹ with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land.     

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).     

Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming

A thermo-alkaline pectate lyase (BliPelA) gene from an alkaliphilic Bacillus licheniformis strain was cloned and overexpressed in Escherichia coli. Mature BliPelA exhibited maximum activity at pH 11 and 70 °C, and demonstrated cleavage capability on a broad range of substrates such as polygalacturonic acid, pectins, and methylated pectins. The highest specific activity, of 320 U mg⁻¹, was towards polygalacturonic acid. Significant ramie (Boehmeria nivea) fiber weight loss (21.5%) was obtained following enzyme treatment and combined enzyme-chemical treatment (29.3%), indicating a high ramie degumming efficiency of BliPelA. The total activity of recombinant BliPelA reached 1450.1 U ml⁻¹ with a productivity of 48.3 U ml⁻¹ h⁻¹ under high-cell-density cultivation with a glycerol exponential feeding strategy for 30 h in 1-l fed-batch fermenter, and 1380.1 U ml⁻¹ with a productivity of 57.5 U ml⁻¹ h⁻¹ after 24 h under constant glucose feeding in a 20-l fermenter using E. coli as the host. The enzyme yields reached 4.5 and 4.3 g l⁻¹ in 1-l and 20-l fed-batch fermenters, respectively, which are higher than those of most reported alkaline Pels. Based on these promising properties and high-level production, BliPelA shows great potential for application in ramie degumming in textile industry.