The effects of salinity on the cellular permeability and cytotoxicity of Heterosigma akashiwo

A laboratory study using the fish‐killing raphidophyte Heterosigma akashiwo was conducted to examine its capability to grow at salinities below oceanic, and to test the perceived relationship between reduced salinities and increased cytotoxicity. A nonaxenic strain of H. akashiwo isolated from the U.S. Pacific Northwest was exposed to a combination of three salinity (32, 20, and 10) and five temperature (14.7°C, 18.4°C, 21.4°C, 24.4°C and 27.8°C) conditions. Our results demonstrate that cell permeability and cytotoxicity are strongly correlated in unialgal cultures of H. akashiwo, which both increased as salinity decreased from 32 to 10. Furthermore, over a broad median range of salinities (10 and 20), neither temperature nor specific growth rate was correlated with cytotoxicity. However, in cultures grown at the salinity of 32, both temperature and specific growth rate were inversely proportional to toxicity; this relationship was likely due to the effect of contamination by an unidentified species of Skeletonema in those cultures. The presence of Skeletonema sp. resulted in a cytotoxic response from H. akashiwo that was greater than the response caused by salinity alone. These laboratory results reveal the capability of H. akashiwo to become more toxic not only at reduced salinities but also in competition with another algal species. Changes in cell permeability in response to salinity may be an acclimation mechanism by which H. akashiwo is able to respond rapidly to different salinities. Furthermore, due to its strong positive correlation with cytotoxicity, cellular permeability is potentially associated with the ichthyotoxic pathway of this raphytophyte.     

INTERSTRAIN VARIABILITY IN PHYSIOLOGY AND GENETICS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) FROM THE WEST COAST OF NORTH AMERICA1

High levels of intraspecific variability are often associated with HAB species, and this variability is likely an important factor in their competitive success. Heterosigma akashiwo (Hada) Hada ex Y. Hara et M. Chihara is an ichthyotoxic raphidophyte capable of forming dense surface‐water blooms in temperate coastal regions throughout the world. We isolated four strains of H. akashiwo from fish‐killing northern Puget Sound blooms in 2006 and 2007. By assessing numerous aspects of biochemistry, physiology, and toxicity, we were able to describe distinct ecotypes that may be related to isolation location, source population, or bloom timing. Contrasting elements among strains were cell size, maximum growth and photosynthesis rates, tolerance of low salinities, amino acid use, and toxicity to the ciliate grazer Strombidinopsis acuminatum (Fauré‐Fremiet). In addition, the rDNA sequences and chloroplast genome of each isolate were examined, and while all rDNA sequences were identical, the chloroplast genome identified differences among the strains that tracked differences in ecotype. H. akashiwo strain 07A, which was isolated from an unusual spring bloom, had a significantly higher maximum potential photosynthesis rate (28.7 pg C · cell^?1 · h^?1) and consistently exhibited the highest growth rates. Strains 06A and 06B were not genetically distinct from one another and were able to grow on the amino acids glutamine and alanine, while the other two strains could not. Strain 07B, which is genetically distinct from the other three strains, exhibited the only nontoxic effect. Thus, molecular tools may support identification, tracking, and prediction of strains and/or ecotypes using distinctive chloroplast gene signatures.     

DESCRIPTION OF VIRIDILOBUS MARINUS (GEN. ET SP. NOV.), A NEW RAPHIDOPHYTE FROM DELAWARE'S INLAND BAYS

Delaware's Inland Bays (DIB), USA, are subject to blooms of potentially harmful raphidophytes, including Heterosigma akashiwo. In 2004, a dense bloom was observed in a low salinity tributary of the DIB. Light microscopy initially suggested that the species was H. akashiwo; however, the cells were smaller than anticipated. 18S rDNA sequences of isolated cultures differed substantially from all raphidophyte sequences in GenBank. Phylogenetic analysis placed it approximately equidistant from Chattonella and Heterosigma with only ~96% sequence homology with either group. Here, we describe this marine raphidophyte as a novel genus and species, Viridilobus marinus (gen. et sp. nov.). We also compared this species with H. akashiwo, because both species are superficially similar with respect to morphology and their ecological niches overlap. V. marinus cells are ovoid to spherical (11.4 × 9.4 μm), and the average number of chloroplasts (4 per cell) is lower than in H. akashiwo (15 per cell). Pigment analysis of V. marinus revealed the presence of fucoxanthin, violaxanthin, and zeaxanthin, which are characteristic of marine raphidophytes within the family Chattonellaceae of the Raphidophyceae. TEM and confocal microscopy, however, revealed diagnostic microscopic and ultrastructural characteristics that distinguish it from other raphidophytes. Chloroplasts were in close association with the nucleus and thylakoids were arranged either parallel or perpendicular to the cell surface. Putative mucocysts were identified, but trichocysts were not observed. These features, along with DNA sequence data, distinguish this species from all other raphidophyte genera within the family Chattonellaceae of the Raphidophyceae.     

The photobiology of Heterosigma akashiwo. Photoacclimation,diurnal periodicity,and its ability to rapidly exploit exposure to high light

Periodic and seasonal exposure to high light is a common occurrence for many near‐shore and estuarine phytoplankton. Rapid acclimatization to shifts in light may provide an axis by which some species of phytoplankton can outcompete other microalgae. Patterns of photoacclimation and photosynthetic capacity in the raphidophyte Heterosigma akashiwo (Hada) Hada ex Hara et Chihara isolated from the mid‐Atlantic of the United States were followed in continuous cultures at low‐ and high‐light intensities, followed by reciprocal shifts to the opposite light level. The maximum quantum yield (F_v/F_m) as well as the photosynthetic cross‐section (σ_PSII) of photosystem II was higher in high‐light cultures compared to low‐light cultures. Significant diurnal variability in photochemistry and photoprotection was noted at both light levels, and high‐light‐acclimated cultures displayed greater variability in photoprotective pathways. When shifted from low to high light, there was only a slight and temporary decline in maximum quantum yield, while cell specific growth more than doubled within 24 h. Rapid acclimation to high light was facilitated by short‐term photoprotection (nonphotochemical quenching), reduced PSII reaction center connectivity, and electron transport. Short‐term increases in de‐epoxidated xanthophyll pigments contributed to nonphotochemical protection, but lagged behind initial increases in nonphotochemical quenching and were not the primary pathway of photoprotection in this alga. By 48 h, photochemistry of cultures shifted from low to high light resembled long‐term high‐light‐acclimated cultures. This isolate of H. akashiwo appears well poised to exploit rapid shifts in light by using unique cellular adjustments in light harvesting and photochemistry.     

CRYPTIC DIVERSITY IN PHYTOPLANKTON CULTURES IS REVEALED USING A SIMPLE PLATING TECHNIQUE1

A simple technique has been developed to probe the occurrence of cryptic genetic diversity in populations of laboratory‐maintained phytoplankton cultures. This agarose‐based method allows the investigator to plate a broad range of fragile algae, including representatives of the Raphidophyceae, Synurophyceae, Prymnesiophyceae, and Dinophyceae. Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara was selected as a model system for our genetic diversity study. Further optimization of the plating technique for this alga demonstrated that colony formation was independent of the physiological state of the parent culture and yet dependent on incident light intensity. The density at which cells were plated affected colony formation and the rate of growth, with intermediate densities (～10³ cells per plate) performing best. High‐metal stress was used as the selective screen for assessing genetic variability within a single H. akashiwo culture. To this end, “clonal” lines (quotation marks used to indicate that within‐clone diversity was expected) were generated from individual plated colonies, and their tolerance was measured by plating on selective medium. Results of these experiments suggest the following: (i) “clonal” lines generated from a single H. akashiwo culture displayed a significant variation in stress tolerance; (ii) “clonal” lines chosen for their ability to grow on selective plates retained this tolerance in the absence of stress, indicating that the observed variation is heritable; and (iii) genetic variation is continually generated in growing cultures. Our results are consistent with a conceptual model, presented here, in which stress tolerance among the individuals in a culture has a genetic basis that varies over a continuous spectrum.     

Incorporating thresholds into understanding salinity tolerance: A study using salt‐tolerant plants in salt marshes

Although salinity in many ecosystems such as salt marshes can be extremely high, an asymmetry in salinity range between experimental studies (relatively narrow) and field conditions (potentially broad) has strongly affected current understanding of plant salinity tolerance. To improve understanding, it is thus important to examine plant tolerances over a broad range of salinities and identify potential tolerance thresholds. We examine tolerances of two widely distributed marsh plants, Suaeda salsa and Salicornia europaea, to salinities ranging from 0 to 100 g/kg, and determine survival, above‐ and belowground biomass after 8 weeks of salinity treatment. Both species, Sa. europaea in particular, have much broader salinity tolerances than other plants previously examined, (2) plant survival, above‐ and belowground biomass have remarkably different responses to salinity, and (3) there is a nonlinear, threshold response of S. salsa to salinity, above which S. salsa survivorship drastically decreases. These results provide multiple important insights. Our study suggests that the potential for using these halophytes to revegetate and restore salt‐affected land may be greater than previously thought, and highlights the importance of studying multiple plant responses. Importantly, our study calls for a better integration of thresholds into understanding plant salinity tolerances and their applications.     

Predator-Induced Fleeing Behaviors in Phytoplankton: A New Mechanism for Harmful Algal Bloom Formation?

In the plankton, heterotrophic microbes encounter and ingest phytoplankton prey, which effectively removes >50% of daily phytoplankton production in the ocean and influences global primary production and biochemical cycling rates. Factors such as size, shape, nutritional value, and presence of chemical deterrents are known to affect predation pressure. Effects of movement behaviors of either predator or prey on predation pressure, and particularly fleeing behaviors in phytoplankton are thus far unknown. Here, we quantified individual 3D movements, population distributions, and survival rates of the toxic phytoplankton species, Heterosigma akashiwo in response to a ciliate predator and predator-derived cues. We observed predator-induced defense behaviors previously unknown for phytoplankton. Modulation of individual phytoplankton movements during and after predator exposure resulted in an effective separation of predator and prey species. The strongest avoidance behaviors were observed when H. akashiwo co-occurred with an actively grazing predator. Predator-induced changes in phytoplankton movements resulted in a reduction in encounter rate and a 3-fold increase in net algal population growth rate. A spatially explicit population model predicted rapid phytoplankton bloom formation only when fleeing behaviors were incorporated. These model predictions reflected field observations of rapid H. akashiwo harmful algal bloom (HAB) formation in the coastal ocean. Our results document a novel behavior in phytoplankton that can significantly reduce predation pressure and suggests a new mechanism for HAB formation. Phytoplankton behaviors that minimize predatory losses, maximize resource acquisition, and alter community composition and distribution patterns could have major implications for our understanding and predictive capacity of marine primary production and biochemical cycling rates.     

Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro-organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima-Nada, the Seto Inland Sea, in 1992 and 1993. In Har-ima-Nada, both flagellates appeared at low levels (< 1 cell mL^?1), and killer micro-organisms against the two flagellates (C-killer for C. antiqua and H-killer for H. akashiwo) also appeared at low densities (< 2 mL^?1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL^?1), and C-killers occurred at a low level (≤ 3.4 mL^?1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H-killers revealed a close relationship with that of H. akashiwo populations. H-killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C-killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro-organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.     

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.     

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

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

Generalist versus specialist strategies of plasticity: snail responses to predators with different foraging modes

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Interactions between a pollinating seed predator and its host plant: the role of environmental context within a population

Plant–insect interactions often are important for plant reproduction, but the outcome of these interactions may vary with environmental context. Pollinating seed predators have positive and negative effects on host plant reproduction, and the interaction outcome is predicted to vary with density or abundance of the partners. We studied the interaction between Silene stellata, an herbaceous perennial, and Hadena ectypa, its specialized pollinating seed predator. Silene stellata is only facultatively dependent upon H. ectypa for pollination because other nocturnal moth co‐pollinators are equally effective at pollen transfer. We hypothesized that for plants without conspecific neighbors, H. ectypa would have higher visitation rates compared to co‐pollinators, and the plants would experience lower levels of H. ectypa pollen deposition. We predicted similar oviposition throughout the study site but greater H. ectypa predation in the area without conspecific neighbors compared to plants embedded in a naturally high density area. We found that H. ectypa had consistently higher visitation than moth co‐pollinators in all host plant contexts. However, H. ectypa pollinator importance declined in areas with low conspecific density because of reduced pollen deposition, resulting in lower seed set. Conversely, oviposition was similar across the study site independent of host plant density. Greater likelihood of very high fruit predation combined with lower pollination by H. ectypa resulted in reduced S. stellata female reproductive success in areas with low conspecific density. Our results demonstrate local context dependency of the outcomes of pollinating seed predator interactions with conspecific host plant density within a population.     

Maintenance of photosynthesis and the antioxidant defence systems have key roles for survival of Halopeplis perfoliata (Amaranthaceae) in a saline environment

• Coastal salt marsh plants employ various combinations of morphological and physiological adaptations to survive under saline conditions. Little information is available on salinity tolerance mechanisms of Halopeplis perfoliata, a C3 stem succulent halophyte.
• We investigated the growth, photosynthesis and antioxidant defence mechanisms of H. perfoliata under saline conditions (0, 150, 300 and 600 mM NaCl) in an open greenhouse.
• Optimal shoot succulence, projected shoot area and relative growth rate were obtained in the low (150 mm NaCl) salinity treatment, while growth was inhibited at the highest salinity (600 mm NaCl). The CO₂ compensation point and carbon isotope composition of biomass confirmed C₃ photosynthesis. Increases in salinity did not affect the photosynthetic pigment content or maximum quantum efficiency of PSII of H. perfoliata. Assimilation of CO₂ (A) also remained unaffected by salinity. A modest effect on some gas exchange and photochemistry parameters was observed at 600 mm NaCl. With increasing salinity, there was a continual increase in respiration, suggesting utilisation of energy to cope with saline conditions. Under 300 and 600 mm NaCl, there was an increase in H₂O₂ and MDA with a concomitant rise in AsA, GR content and CAT activity.
• Hence, H. perfoliata appears to be an obligate halophyte that can grow up to seawater salinities by modulating photosynthetic gas exchange, photochemistry and the antioxidant defence systems.

     

Can protective attributes of artificial refuges offset predation risk in lizards?

Artificial refuges are often used to supplement habitat in areas where natural shelters have been degraded or removed. Although artificial refuges are intended to support particular species, they may be equally attractive and accessible to others, including predators. We explored the influence of snake predation risk and shelter attributes on the overnight use of different artificial refuges (timber, tiles, and iron) using the predator‐prey relationship between Boulenger's skink, Morethia boulengeri and the curl snake, Suta suta. We collected adult M. boulengeri from two bioregions in south‐eastern Australia: the Riverina, where the two species co‐occur, and the South Western Slopes, where S. suta does not occur. Two adult S. suta were collected for use as chemical donors. We conducted four experiments on overnight refuge choice to determine: (i) predator‐scent avoidance, (ii) artificial refuge preferences, (iii) a trade‐off between a preferred refuge and predator‐avoidance, and (iv) the effect of gap height on refuge preference. We found that skinks avoided predator‐scented refuges in favour of identical, but unscented refuges. Skinks preferred timber refuges, and most skinks maintained this preference when predator‐scent was added. However, when gap height was manipulated, skinks shifted to the refuge with the smallest gap. Skinks displayed complex regional variation in behaviour; skinks from both bioregions avoided predator‐scent, but in the trade‐off experiment, skinks from the South Western Slopes were less likely to avoid predator‐scented timber refuges than those from the Riverina. Our findings suggest that protective refuge attributes, such as small gap height, can offset the risk implied by predator‐scent within a refuge. This study highlights the need to consider predator‐prey interactions when designing and using artificial refuges for habitat restoration or biological monitoring.     

Effect of salinity on the distribution, growth, and toxicity of Karenia spp.

The first recorded bloom of Karenia spp., resulting in brevetoxin in oysters, in the low salinity waters of the Northern Gulf of Mexico (NGOMEX) occurred in November 1996. It raised questions about the salinity tolerance of Karenia spp., previously considered unlikely to occur at salinities <24 psu, and the likelihood that the bloom would reoccur in the NGOMEX. Salinity was investigated as a factor controlling Karenia spp. abundance in the field, using data from the NGOMEX 1996 bloom and Florida coastal waters from 1954 to 2004, and growth and toxin production in cultures of Karenia brevis (Davis) G. Hansen and Moestrup. During the NGOMEX bloom, Karenia spp. occurred much more frequently at low salinities than in Florida coastal waters over the last 50 years. The data suggest that the NGOMEX bloom started on the NW Florida Shelf, an area with a higher frequency of Karenia spp. at low salinities than the rest of Florida, and was transported by an unusual westward surface current caused by Tropical Storm Josephine. The minimum salinity at which growth occurred in culture ranged between 17.5 and 20 psu, but the optimal salinity ranged between low values of 20 or 25 and high values of 37.5–45 psu, depending on the clone. The effect of salinity on toxin production in one clone of K. brevis was complex, but at all salinities brevetoxin levels were highest during the stationary growth phase, suggesting that aging, high density blooms may pose the greatest public health threat. The results demonstrate that Karenia spp. can be a public health threat in low salinity areas, but the risk in the NGOMEX is relatively low. No bloom has occurred since the 1996 event, which was probably associated with a special set of conditions: a bloom along the Florida Panhandle and a tropical storm with a track that set up a westward current.     

Detection of Heterosigma akashiwo (Hada) using specific RNA probes: Variability of RNA content with environmental conditions

The raphidophyte Heterosigma akashiwo, which forms toxic blooms, causes major economical losses to the fish industry because of the fish kills involved. It is therefore important to be able to detect not only H. akashiwo but other toxic phytoplankton species as well, rapidly and accurately to reduce losses by fish kills. With this purpose, DNA sequences from H. akashiwo 18S and 28S rRNA gene regions were studied in silico to design species-specific probes to be used in a microarray format. Three strains of H. akashiwo (AC 265, AC 266 and GUMACC 120) were grown at optimal conditions and transferred into new environmental conditions changing either the light intensity, salinity, temperature or nutrient concentrations, to check if any of these environmental conditions induced changes in the cellular RNA concentration. The aim of this experiment was the calibration of several species-specific probes for the quantification of H. akashiwo. Differences on RNA content were not significant (p < 0.05) in any of the treatments, therefore the calibration curves were validated. The designed probes are reliable for the detection and quantification of H. akashiwo cells in natural waters.     

Climate change and trophic interactions in model temporary pond systems: the effects of high temperature on predation rate depend on prey size and density

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A parasite's modification of host behavior reduces predation on its host

Parasite modification of host behavior is common, and the literature is dominated by demonstrations of enhanced predation on parasitized prey resulting in transmission of parasites to their next host. We present a case in which predation on parasitized prey is reduced. Despite theoretical modeling suggesting that this phenomenon should be common, it has been reported in only a few host–parasite–predator systems. Using a system of gregarine endosymbionts in host mosquitoes, we designed experiments to compare the vulnerability of parasitized and unparasitized mosquito larvae to predation by obligate predatory mosquito larvae and then compared behavioral features known to change in the presence of predatory cues. We exposed Aedes triseriatus larvae to the parasite Ascogregarina barretti and the predator Toxohrynchites rutilus and assessed larval mortality rate under each treatment condition. Further, we assessed behavioral differences in larvae due to infection and predation stimuli by recording larvae and scoring behaviors and positions within microcosms. Infection with gregarines reduced cohort mortality in the presence of the predator, but the parasite did not affect mortality alone. Further, infection by parasites altered behavior such that infected hosts thrashed less frequently than uninfected hosts and were found more frequently on or in a refuge within the microcosm. By reducing predation on their host, gregarines may be acting as mutualists in the presence of predation on their hosts. These results illustrate a higher‐order interaction, in which a relationship between a species pair (host–endosymbiont or predator–prey) is altered by the presence of a third species.