Sinking of Heterosigma Akashiwo Results in Increased Toxicity of This Harmful Algal

Home , Heterosigma akashiwo, Raphidophyte

Harmful Algae 13 (2012) 95–104

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Harmful Algae

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Sinking of Heterosigma akashiwo results in increased toxicity of this harmful algal

bloom species

a a a,b,

Lucas Powers , Irena F. Creed , Charles G. Trick *

Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada

Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5B7, Canada

A R T I C L E I N F O A B S T R A C T

Article history: Notable physiological responses such as toxicity and sinking rates of the red tide forming raphidophyte

Received 7 June 2010

Heterosigma akashiwo are correlated with high levels of macronutrient stress. Individual cells of this

Received in revised form 21 September 2011

species are also capable of forming benthic vegetative cysts that overwinter in marine sediment and

Accepted 12 October 2011

contribute to bloom propagation in subsequent seasons. It was hypothesized that there is variability in

Available online 22 October 2011

the rates of sinking within cell cultures and that sinking cells are more toxic than the neutrally buoyant or

ﬂoating cells. Using laboratory-based settling columns, various isolates of H. akashiwo were allowed to

Keywords:

separate, and the toxicities of sinking and ﬂoating populations were analyzed. Sinking and ﬂoating rates

Heterosigma akashiwo

were signiﬁcantly higher during the late stationary growth phase for all isolates. For two H. akashiwo

Sinking

Bioassays isolates, sinking populations were signiﬁcantly more toxic than those that were positively buoyant. A

Toxicity similar trend was observed in a third strain, however the relationship was not signiﬁcant. Differences in

Bloom propagation adaptive ecophysiology among the different strain likely caused the variation. It is suggested that the

most toxic cells within a bloom are those found at the lower depths, potentially interacting with the

benthic community or ensuring that subsequent bloom propagation contains cells with the potential for

toxicity.

1. Introduction extremely dense cellular aggregations, which creates a physical

barrier to light penetration that subsequently results in anoxic

The global prevalence and severity of harmful algal blooms conditions as blooms dissipate; (ii) producing potent toxins that

(HABs) appears to be increasing in many marine ecosystems bioaccumulate and are known to be directly harmful to humans;

(Smayda, 1989; Horner et al., 1997; Anderson et al., 2002; Heisler and (iii) demonstrating allelopathic and anti-biological strategies

et al., 2008). HABs are characterized by the dominance of a single that can induce high mortality in a wide range of aquatic

phytoplankton species, eventually forming dense concentrations organisms, particularly ﬁsh, with devastating effects on aquacul-

of algal biomass that threaten the health of the ecosystem by a ture (Honjo, 1993). Considerable debate surrounds species in this

number of mechanisms. It remains unclear, however, precisely third category, as no single process or mechanism has been

what environmental parameters mediate bloom formation and conclusively identiﬁed to explain how these species achieve

what toxicological mechanisms HAB species employ that result in ichthyotoxicity.

these harmful effects (Anderson et al., 2002; Hallegraeff and Hara, A phytoplankton species of particular concern within this third

2003), with considerable variation among the relatively small category is the red-tide forming alga, Heterosigma akashiwo (Hada)

percentage of algal genera capable of forming HABs (Morris, 1999; (Hara and Chihara, 1987). Coastal blooms of this ﬁsh-killing

Landsberg, 2002). raphidophyte have been observed in both the Atlantic and Paciﬁc

Generally, potentially harmful species can affect a community Oceans and have been implicated in ﬁsh-kills in aquaculture

in one of the following ways (Smayda, 1997a,b; Hallegraeff and operations in Canada, Chile, Japan and New Zealand (Hallegraeff and

Hara, 2003): (i) causing signiﬁcant water discoloration due to Hara, 2003). Additionally, severe economic losses associated with

ﬁn-ﬁsh mortality attributed to H. akashiwo have been reported in

Japan (Honjo, 1993), British Colombia, Canada (Taylor, 1991; Taylor

and Haigh, 1993), Washington State, USA (Connell and Cattolico,

* Corresponding author at: Department of Biology, University of Western 1996), and New Zealand (Chang et al., 1990). More recently, blooms

Ontario, London, Ontario N6A 5B7, Canada. Tel.: +1 519 661 3899;

of H. akashiwo have been reported in San Francisco Bay, USA

fax: +1 519 850 2343.

(Herndon and Cochlan, 2006) and the inner waters of British

E-mail addresses: [email protected] (L. Powers), [email protected] (I.F. Creed),

[email protected] (C.G. Trick). Colombia and Washington State (Horner et al., 1997).

96 L. Powers et al. / Harmful Algae 13 (2012) 95–104

Although a global problem, the mechanism of H. akashiwo If resource conditions, in particular the availability of macro-

toxicity has yet to be determined. Because H. akashiwo is capable of nutrients, positively affect both the sinking rates and potential

harming a wide spectrum of marine organisms such as zooplank- toxicity of a cell, then it is likely that the most highly toxic cells will

ton, copepods, benthic larvae and ﬁsh, the toxin(s) or mechanisms be found lower in the water column because both are common

responsible for its potent toxicity remain elusive. Studies have phytoplankton responses to macronutrient stress. There are

attributed the toxicity associated with raphidophytes to the notable ecological and practical consequences associated with

production of two reactive oxygen species (ROS), superoxide (O2 ) this possibility. If more highly toxic cells migrate to lower depths, it

(Oda et al., 1998; Marshall et al., 2005) and hydrogen peroxide increases the possibility that these cells will aggregate and

(H2O2) (Twiner and Trick, 2000; Twiner, 2002); haemolytic overwinter in sediment in a vegetative state (Kiorboe et al.,

compounds, which lyse red blood cells (de Boer et al., 2004; Fu 1994). Individuals that survive in marine sediment seed the

et al., 2004; Ling and Trick, 2010); and a brevetoxin-like organic following year’s potential bloom, and thus if highly toxic cells sink

neurotoxin (Khan et al., 2008). However, none of these putative and overwinter preferentially, then regions may become more

toxic agents have been independently linked to the ichthyotoxicity prone to toxic blooms over time. The purpose of this research was

of H. akashiwo. Finﬁsh may not be the target of H. akashiwo toxicity, to establish the association between cellular nutrient status,

but rather they may be negatively affected by compounds sinking and toxicity. Speciﬁcally, under what conditions and at

produced by H. akashiwo as a means of bloom formation, what point in its cellular life cycle is Heterosigma most prone to

continuance or maintenance. sinking? Additionally, what is the toxic potential of cells under

Blooms of raphidophyte HAB species, such as Heterosigma, but various nutrient stresses, and how is toxicity related to the

also Chattonella, often appear to be ephemeral, because the cells physiological responses of sinking and ﬂoating? This knowledge is

disperse or concentrate to the highly visible surface bloom vital in establishing an increased predictive power of toxic bloom

(Hershberger et al., 1997). The dynamics of this vertical formation and also in understanding the spatial dynamics of toxic

concentration is often critical to the energetic and nutrient needs blooms in natural environments. In a broad sense, the ultimate

of the migrating cells and these dynamics have drawn considerable goal of this HAB research is to develop methodologies for

research attention. From early studies (Smayda, 1970, 1997a) it has effectively mediating the harmful effects of H. akashiwo and

become established that ﬂagellated cells such as H. akashiwo can possibly preventing their proliferation in marine coastal waters.

regulate its relative position via both active and passive locomo-

tion processes to optimize access to resources such as nutrients 2. Materials and methods

and light, a critical feature in photoreceptive strategy.

Resource availability also regulates the formation of cysts, a life 2.1. Phytoplankton cultures and nutrient conditions

cycle stage that is relatively rare among HAB species (Smayda,

1997b). Cysts generally form after the dissipation of high biomass All phytoplankton strains were non-axenic and grown in batch

blooms, when nutrient stress becomes physiologically over- cultures in autoclave-sterilized, modiﬁed ESAW medium (Harrison

whelming for the cells or cooler temperatures signal the imminent et al., 1980). A 10% (v/v) inoculum of cells was added to 45 mL of

onset of winter conditions. Vegetative cysts are extremely hardy medium to a ﬁnal volume of 50 mL in autoclaved 250 mL

and are capable of overwintering in the benthic sediment and Erlenmeyer ﬂasks. Cells were acclimated to the experimental

reemerging as reproductive cells during the following spring and N:P ratio for two generations under constant temperature (18 8C)

1 1

summer seasons (Tomas, 1978). H. akashiwo cysts sink rapidly to and irradiance (160 mmol photons m s ) prior to the start of the

the sediment and are thus less likely to senesce due to experiment (experimental T0). Since light:dark cycles could induce

environmental stresses or are consumed by an algal grazer before a diel migration cycle, all cultures were maintained under a

1 1

depositing into the benthic ecosystem (Smayda, 1990, 1998). constant light ﬂux of 160 mmol photons m s . Balanced growth

Furthermore, cells that do re-emerge after overwintering can also conditions, representative of nitrate to phosphate ratios found in

be toxic towards benthic algal grazers, and thus the potential natural marine ecosystems not under environmental stress, were

effects of H. akashiwo are not limited to the pelagic environment characterized by a 15:1 nitrate to phosphate molar ratio in the

(Tomas and Deason, 1981). modiﬁed ESAW media. Phosphate-limited media were character-

Not all blooms of H. akashiwo are toxic and not all toxic blooms ized by a 50:1 nitrate to phosphate molar ratio, achieved by

remain toxic at all times; blooms may exhibit traits of potent increasing the concentration of nitrate in ESAW media. Nitrate-

toxicity or remain relatively harmless in a given year (Black, limited media contained a molar nitrate to phosphate ratio of 5:1.

2000). H. akashiwo grown in nutrient-limited conditions generally Three strains of H. akashiwo, strain Can 764R (Northeast Paciﬁc

exhibits greater toxicity due to changes in physiological dynamics Culture Collection), strain ‘‘Can Ed’’ (from coastal British Columbia

caused by the stress of severe nutrient depletion over time. The waters, isolated by Dr. Ed Black, Department of Fisheries and

change in toxicity is often related to the changing nutrient Oceans, Canada) and strain ‘‘Spain’’ (isolated by A. Butron from

environment, with nutrient stressed cells being more toxic than waters of the Basque Region, Spain in 2002), were compared in this

the nutrient replete cells in a population (Honjo, 1993). In the life study. Tetraselmis apiculata (UTEX 2562), a strain commonly used

cycle of the bloom there are complex simultaneous activities as a food source in aquaculture facilities and considered to be non-

(Smayda, 1997a). For example, the accumulation of biomass toxic, was used as a control species, given that it is the same

eventually becomes self-shading and/or nutrient limiting, and approximate size and shape of the Heterosigma isolates.

thus individuals may then employ variable strategies to cope with

these stresses. The result is an accumulation of cells in the surface 2.2. Culture growth and growth phases

waters with other individuals sinking to the benthos. How an

individual cell behaves will depend on the greatest stress to that Beginning on the day of inoculation, all cultures were sampled

particular cell and where nutrients are within the water column. every other day to monitor cell growth. A 0.5 mL subsample of

These are not independent because nutrient-limited cells often culture sample was pipetted into 8 mL FloTubes in a sterile

sink within the water column (Bienfang et al., 1982; Lecourt et al., laminar ﬂow hood and analyzed for cell counts on a FACSCalibur

1996; Muggli et al., 1996). This adds a spatial element when ﬂow cytometer [B-D Biosciences]. Samples were run for one

considering the dynamics of cells and toxins in a harmful algal minute using a previously formatted marine phytoplankton

bloom. template. Monitoring continued until cell counts ceased to

L. Powers et al. / Harmful Algae 13 (2012) 95–104 97

increase for at least two or at most three samplings. After at least generated using the following equation:

two samplings with no observed increase, the culture was

considered to be in late stationary growth phase (after approxi- BfracðobsÞ BfracðpredÞ h

A or c ¼ (1)

mately 14–16 days). Early stationary phase was delineated as the Btot t

ﬁrst day that exponential growth of cultures was observed to stop

(after approximately 10–12 days). Exponential growth phase was

where Bfrac(obs) is the observed biomass in the top (ﬂoating) or

characterized by exponential increases in cell numbers (approxi-

bottom (sinking) fractions, Bfrac(pred) is the predicted biomass of the

mately 8–10 days).

fraction based on the sum of biomass in the column multiplied by

the fraction volume relative to the total volume; h is the height of

2.3. Column fractionations

the column of water, and t is the incubation time in hours.

Settling column experiments were conducted during the

2.6. Toxicity bioassay

exponential growth phase, early stationary growth phase, and

late stationary growth phase. Once in the desired growth phase,

Each sinking cell and ﬂoating cell fraction from each column

quadruplicate ﬂasks of 50 mL were pooled together into a single

was used in a standard bioassay to assess the relative toxicity of

250 mL Erlenmeyer ﬂask for each culture. Culture (150 mL) was

cells found within the fractions. A sample of cells from each

then poured into a corresponding borosilicate settling column, 1

fraction was standardized to a concentration of 15,000 cells mL .

each 53 cm high, 3.5 cm in diameter with a 250 mL total capacity.

A standardized sample (1 mL) was added to each well on a 24-well

Each column was covered in industrial grade aluminum foil so that

tray and left to incubate with ten young (1-day old) or ten mature

no light penetrated the columns. Separations were conducted for a

(2-days old) brine shrimp per well (Artemia salina) (Vanhaecke

6-h period, in darkness and at a constant temperature of 18 8C.

et al., 1981). Every fraction was assayed with both young and

After each 6-h-settling period, three distinct fractions of the

mature brine shrimp independently. Addition of HCl (20%, v/v) was

column sample were removed. The top 25 mL was removed using

used as a positive control, and these samples were incubated with

gentle aspiration from above, collecting all media to a prescribed

brine shrimp under identical conditions to culture samples.

mark. The top sample was representative of the ﬂoating cell

Autoclave-sterilized modiﬁed ESAW media (salt solution only,

fraction. The bottom 25 mL of the undisturbed column, represen-

no macronutrients or trace elements added) were used as negative

tative of the sinking cell fraction, was slowly decanted from a

controls and were treated identically to other treatments. After the

bottom drain. The intermediate fraction consisted of the remaining

initial 24-h incubation, brine shrimp were observed under a

100 mL and was representative of cells without a tendency to ﬂoat

microscope and classiﬁed as dead, struggling, or alive. Following

or sink. Once all fractions were separated, 0.5 mL subsamples were

the initial count, brine shrimp were incubated for a subsequent 24-

removed from each fraction for cell counts. Additionally, 15 mL

h period and dead, struggling and healthy brine shrimp were

subsamples were collected from each respective fraction to be

tallied again. Struggling individuals were readily distinguishable

ﬁltered for chlorophyll-a based biomass analysis.

from dead and healthy brine shrimp; struggling individuals lacked

coordination in movement and/or feeding. Extended observations

2.4. Biomass measurements

(72 h) indicated that struggling individuals regained a normal

motility behavior, rather than succumb to the toxicity of the

The total biomass of each fraction for all isolates during assessment.

experiments was quantiﬁed using an extracted chlorophyll

method (Parsons et al., 1984). Exactly 15 mL of fraction samples

2.7. Statistical analysis

were ﬁltered over 25 mm diameter, 0.4 mm pore polycarbonate

ﬁlters using a vacuum ﬁltering apparatus. Filters were then placed

All data were analyzed using SPSS 17.0 statistical software.

in 90% acetone and transferred to a 4 8C freezer for 24 h. After that

Floating and sinking rates were compared across growth phases

period, ﬁlters were removed from the acetone using forceps and

and between nutrient treatments using one-way ANOVAs. All

the remaining solution was analyzed on a Turner-10AU Fluorom-

brine shrimp bioassay mortality data were analyzed using one-

eter according to the protocol outlined in Welschmeyer (1994).

and two-way ANOVAs and were checked for homogeneity and

Raw ﬂuorescence output was transformed into chlorophyll-a

independent factor interactions. An alpha of 0.05 was used in all

concentrations (mg mL ) using a standard curve generated by

statistical analyses. Toxicity bioassay results were computed and

analyzing spinach extracts of known concentrations on a

compared between isolates using values that combined the total

spectrophotometer at wavelengths of 630 nm, 647 nm, 664 nm

number of brine shrimp observed to be dead and the total number

and 750 nm.

observed to be struggling. These ‘‘dead + struggling’’ values

permitted greater statistical power and increased relative toxicity

2.5. Floating and sinking rates

differences among isolates. Means are reported 1 SD.

Floating and sinking rates of populations are mechanistically

deﬁned and cannot be directly related to the physiological 3. Results

motivation of the cells. For example, cells expressing ascending

vertical migration would be pooled with cells that are non- 3.1. Floating and sinking rates

committal to migration but more buoyant than the medium.

Similarly, cells expressing descending vertical migration would be The fractionation of the culture into sinking and ﬂoating sub-

pooled with dense cells accumulated through passive sinking. For populations occurred in all strains studied. Even though H.

these reasons, the relative distribution differences in the stable akashiwo is recognized as a surface forming bloom species that

experimental column was used to define the floating and sinking forms sinking cysts (Honjo, 1993), the sinking and floating rates of

rates of the populations within each treatment. Relative ﬂoating the comparison ﬂagellate (T. apiculata strain 2562.) equaled or

and sinking rates for each culture were calculated using a modiﬁed exceeded that of H. akashiwo strains. Under these conditions, the

SETCOL method (Bienfang et al., 1982). Once the biomass of each physiological or ecological importance of sinking and/or ﬂoating to

fraction was known, ﬂoating (A) and sinking (c) rates were H. akashiwo was not distinctive.

98 L. Powers et al. / Harmful Algae 13 (2012) 95–104

Fig. 1. Floating rates (m d ) of all isolate cultures in late stationary, early stationary, and exponential growth phases grown under phosphate limited conditions (50 nitrate:1

phosphate) and nitrate limited conditions (5 nitrate:1 phosphate). Floating rates were calculated after a 6 h settling duration using chlorophyll-a as a proxy for biomass in the

modiﬁed SETCOL equation.

The rates of sinking and ﬂoating were most strongly inﬂuenced separated into two sub-populations with dynamic sinking and

by two factors: the strain being studied (Figs. 1 and 2) and the ﬂoating rates. For every isolate examined, as well as the control

growth phase (Tables 1 and 2). One strain, H. akashiwo strain ED, species, the greatest ﬂoating rates and sinking rates were observed

exhibited little tendency to partition into a sinking or ﬂoating during late stationary growth phase (Figs. 1 and 2). Floating and

subpopulations. While the H. akashiwo strain Spain readily sinking rates during the late stationary phase were signiﬁcantly

Fig. 2. Sinking rates (m d ) of all isolate cultures in late stationary, early stationary, and exponential growth phases grown under phosphate limited conditions (50:1) and

nitrate limited conditions (5:1). Floating rates were calculated after a 6 h settling duration using chlorophyll-a as a proxy for biomass in the modiﬁed SETCOL equation.

L. Powers et al. / Harmful Algae 13 (2012) 95–104 99

Table 1

Median ﬂoating rates of all three H. akashiwo isolates and T. apiculata 2562 (m d ) (25% quartile values). Sinking rates denoted with the same lower case letter were not

signiﬁcantly different from each other. Median sinking rates were calculated by determining the median values for all isolates during each respective growth phase.

Growth phase Median ﬂoating rate (m d ) 25% quartile value 75% quartile value p-Value

Late stationary 0.422 a 0.195 0.645 p < 0.05

Early stationary 0.033 b 0.008 0.060 p > 0.05

Exponential 0.049 b 0.019 0.090

Mean values shown with the same lower-case letter were not signiﬁcantly different.

Table 2

Median sinking rates of all three H. akashiwo isolates and T. apiculata 2562 (m d ) (25% quartile values). Sinking rates denoted with the same lower case letter were not

signiﬁcantly different from each other. Median sinking rates were calculated by determining the median values for all isolates during each respective growth phase.

Growth phase Median Sinking rate (m d ) 25% quartile value 75% quartile value p-Value

Late stationary 0.871 a 0.553 1.218 p < 0.05

Early stationary 0.159 b 0.140 0.215 p > 0.05

Exponential 0.174 b 0.174 0.137

Mean values shown with the same lower-case letter were not signiﬁcantly different.

different from those observed in both the exponential growth The sinking population of H. akashiwo strain Can Ed was

phase and early stationary growth phase for each isolate of H. signiﬁcantly more toxic than ﬂoating population under both

akashiwo and T. apiculata 2562. Within the late stationary growth nutrient conditions in late stationary growth phase (p < 0.001).

phase, nutrient regime (phosphorus (P)-limited, 50 nitrate:1 There was also a signiﬁcant difference in the toxicity of H. akashiwo

phosphate; and nitrogen (N)-limited, 5 nitrate:1 phosphate) did strain Can Ed between respective nutrient conditions (p < 0.001).

not signiﬁcantly affect ﬂoating and sinking rates for most of the H. Floating populations of H. akashiwo strain Can Ed grown under P-

akashiwo isolates or the control species (p > 0.05). The one limited conditions (50 nitrate:1 phosphate) displayed the least

exception was H. akashiwo strain Can 764R where the sinking toxicity of any isolate and fraction during late stationary growth

rate of P-limited, late stationary cells was about 3 more rapid phase, negatively affecting (mortality + struggling individuals)

than similarly aged N-limited cells. Even with the large variation in only 8.8(7.2)% of brine shrimp.

the strain dynamics, within any one isolate the sinking rate was The response of H. akashiwo strain Spain was more variable,

consistently in the order of 2 faster than the floating rates. with a significant difference in relative toxicity between floating

populations and sinking populations within both nutrient condi-

3.2. Toxicity bioassays tions (p < 0.001), but no signiﬁcant difference between P-limited

and N-limited conditions (p = 0.678). Sinking populations of H.

Each strain of H. akashiwo was signiﬁcantly more toxic to A. akashiwo strain Spain grown under N-limited conditions displayed

salina than the control strain T. apiculata 2562 at all nutrient the greatest toxicity of any isolate and fraction during late

conditions and during all growth phases (p < 0.05) (Table 3). T. stationary growth phase, negatively affecting 43.1(16.7)%.

apiculata 2562 showed no indication of toxicity towards A. salina. Further, no signiﬁcant differences in relative toxicity between

Additionally, there was no signiﬁcant difference between the ﬂoating and sinking populations were found for H. akashiwo strain

relative toxicities of each H. akashiwo strain (p > 0.05). Likewise, no Can 764R within each nutrient condition (p = 0.134), nor between

signiﬁcant difference in toxicity was observed between P-limited respective nutrient conditions (p = 0.996). Although differences

and N-limited conditions for all three H. akashiwo strains were not signiﬁcant, there was a clear trend of sinking populations

(p > 0.05). being more toxic than ﬂoating populations grown at N-limited

Table 3

Mean number of affected brine shrimp (Artemia salina) for ﬂoating and sinking fractions of all isolates of H. akashiwo and the biological control T. apiculata 2562 during late

stationary growth phase (SD).

Isolate Nitrate to phosphate ratio Fraction Mean dead + struggling A. salina per 10 individuals SD p-Value

Can Ed 50:1 Floating 0.88 a 0.72 <0.001

Sinking 2.31 b 1.45

5:1 Floating 2.12 a 1.21 <0.001

Sinking 3.60 b 1.40

Spain 50:1 Floating 3.13 a 1.54 <0.001

Sinking 4.40 b 1.54

5:1 Floating 2.90 a 1.21 <0.001

Sinking 4.31 b 1.67

Can 764R 50:1 Floating 3.50 a 1.60 0.134

Sinking 3.50 a 1.31

5:1 Floating 2.94 a 1.39 0.134

Sinking 4.13 a 1.54

T. apiculata 2562 50:1 Floating 0.06 a 0.12 0.557

Sinking 0.13 a 0.34

5:1 Floating 0.00 a 0.00 0.557

Sinking 0.00 a 0.00

Mean values shown with the same lower-case letter within each N:P ratio were not signiﬁcantly different.

100 L. Powers et al. / Harmful Algae 13 (2012) 95–104

Fig. 3. Toxicity bioassay results for H. akashiwo Ed during exponential, early, and

Fig. 4. Toxicity bioassay results for H. akashiwo Spain 2002 during exponential,

late stationary growth phases shown as a percentage of total brine shrimp (SD).

early, and late stationary growth phases shown as a percentage of total brine shrimp

‘‘Upper’’ denotes ﬂoating cell fraction, while ‘Lower’ denotes sinking cell fraction.

(SD). ‘‘Upper’’ denotes ﬂoating cell fraction, while ‘Lower’ denotes sinking cell

Mortalities denoted with the same lower case letter were not signiﬁcantly different.

fraction. Mortalities denoted with the same lower case letter were not signiﬁcantly

Brine shrimp were tallied as dead, struggling or alive after 24 h, and again after 48 h.

different. Brine shrimp were tallied as dead, struggling or alive after 24 h, and again

Results shown here represent a 48-h incubation, and SD was calculated by combining

after 48 h. Results shown here represent a 48-h incubation, and SD was calculated by

‘Dead’ and ‘Struggling’ into a single value.

combining ‘Dead’ and ‘Struggling’ into a single value.

conditions; however, this trend was not clear at P-limited growth and the early stage of stationary growth. In none of the

conditions (Figs. 3–6). isolates did toxicity increase with prolonged nutrient starvation. H.

No signiﬁcant interactions between nutrient condition and akashiwo strain Can Ed was most toxic during the early stationary

fraction (sinking/ﬂoating populations) were observed for any phase under N-limiting conditions. H. akashiwo strain Spain

isolate of H. akashiwo or T. apiculata 2562 during the late stationary achieved its most potent toxicity during the exponential growth

growth phase (p < 0.05). Further, no signiﬁcant differences were phase, also grown under nitrate-limited conditions. Finally, H.

found in the number of affected brine shrimp between young (48-h akashiwo strain Can 764R displayed its highest toxicity during the

old) and juvenile (24-h old) (p < 0.05). Likewise, length of exponential phase under N-limited conditions.

incubation time of brine shrimp with H. akashiwo isolates had

no signiﬁcant effect on the number of affected brine shrimp for 4. Discussion

both juvenile and mature brine shrimp bioassays.

All three strains of H. akashiwo displayed their highest toxicity The fundamental purpose of this research was to examine how

during the transition period between nutrient-replete exponential growth phase and nutrient conditions affect the ﬂoating and

L. Powers et al. / Harmful Algae 13 (2012) 95–104 101

Fig. 5. Toxicity bioassay results for H. akashiwo 764R during exponential, early, and

late stationary growth phases shown as a percentage of total brine shrimp (SD). Fig. 6. Toxicity bioassay results for T. apiculata 2562 during exponential, early, and

‘‘Upper’’ denotes ﬂoating cell fraction, while ‘Lower’ denotes sinking cell fraction. late stationary growth phases shown as a percentage of total brine shrimp (SD).

Mortalities denoted with the same lower case letter were not signiﬁcantly different. ‘‘Upper’’ denotes ﬂoating cell fraction, while ‘Lower’ denotes sinking cell fraction.

Brine shrimp were tallied as dead, struggling or alive after 24 h, and again after 48 h. Mortalities denoted with the same lower case letter were not signiﬁcantly different.

Results shown here represent a 48-h incubation, and SD was calculated by combining Brine shrimp were tallied as dead, struggling or alive after 24 h, and again after 48 h.

‘Dead’ and ‘Struggling’ into a single value. Results shown here represent a 48-h incubation, and SD was calculated by combining

‘Dead’ and ‘Struggling’ into a single value.

sinking behavior of H. akashiwo. It is congruent with the H. communities and laboratory cultures have metabolized all of

akashiwo cellular life cycle that ﬂoating and sinking rates were the nutrients that facilitated exponential growth. Thus cells at this

generally greatest during late stationary growth phase. Under late phase have (i) been exposed to a nutrient-limited environment for

stationary conditions the sinking rate predominated at about 2- a notable duration of time and (ii) stopped allocating metabolic

times the ﬂoating rate. Yet, ascending cells were also enhanced energy resources to cellular growth and will subsequently allocate

during this nutrient-stressed phase. Motility exhibited in ﬂagellate nutrient reserves to cell maintenance. Severe physiological stress

HAB species is a measurable response that results from a complex and the potential to allocate remaining energetic complexes to

interaction between physiological processes intended to maximize motility in order to escape that physiological stress in the water

light receptivity and nutrient uptake and external stresses applied column can explain why sinking rates were greatest and about 2

by the physical environment (Watanabe et al., 1988; Kamykowski ﬂoating rates in the late stationary phase (Grane´li and Johansson,

et al., 1992). An increase in motility has been correlated with 2003), but ﬂoating rates were also signiﬁcantly higher in the late

increasing nutrient depletion as resources become more limited. stationary phase when compared to either of the two previous

In the late stationary growth phase, natural phytoplankton growth phases (Smayda, 1997b).

102 L. Powers et al. / Harmful Algae 13 (2012) 95–104

Floating and sinking rates observed during column fractiona- to increase the potential N-pool in environments with limited

tions for H. akashiwo isolates ranged from about 0.2 to 1.9 m d . supply of inorganic-N.

There is scarce literature directly reporting settling rates in the This model of toxicity is supported by the ﬁnding that the N-

species and isolates examined. Some studies have focused on the limited condition (5 nitrate:1 phosphate) was signiﬁcantly more

settling dynamics of diatom species, where reported settling rates toxic than the phosphate (P)-limited condition (50 nitrate:1

are generally higher, likely due to the fact that individual cells in phosphate) during the growth phase of highest overall toxicity for

many diatom species are often quite large, ranging from 2 to H. akashiwo strains Can Ed and Can 764R (early stationary and

200 mm, and have highly dense silica frustules. In comparison, the exponential, respectively). In contrast, the toxicity of H. akashiwo

‘‘cell wall-free’’ H. akashiwo reaches a maximum size of 15– strain Spain was more affected by P-limitation, illustrating that

25 mm (Smayda, 1970). Bienfang (1980) found that sinking rates in clonal variation is not uncommon in H. akashiwo studies, and this

open ocean phytoplankton communities were signiﬁcantly higher result is consistent with results reported by Furnas (1990) and

when those communities consisted of individuals >20 mm in Martinez et al. (2010).

length, as opposed to communities consisting of small individuals If hemolytic scavenging is the primary mechanism of toxicity in

3–20 mm in length. Based on these previous studies, the ﬂoating H. akashiwo, it is likely that relative toxicities were greatest during

and sinking rates seen during this experiment appear to represent exponential and early stationary growth phases because cells are

realistic rates for natural H. akashiwo communities. However, allocating the majority of energy resources to cell growth and

maximum ﬂoating and sinking rates, however, did not correlate reproduction during the exponential phase. These processes have

with the periods of greatest relative toxicity. In most cases, the only recently slowed or ceased during the early stationary phase,

cells were most toxic against brine shrimp during the actively with some cells still reproducing, thus individuals will attempt to

growing or early stationary phases. maximize nutrient uptake and also have the cellular resources to

During these earlier life cycle phases, the characteristics of actively compete for limited nutrients. Additionally, cells may be

H. akashiwo toxicity were revealed in the A. salina assay. In optimizing the intracellular storage of nutrients before nutrient

contrast to other marine biotoxins [e.g., domoic acid, (Bates and availability becomes so scarce that such processes become

Trainer, 2006)], cell toxicity decreased as cells underwent energetically inefﬁcient (Watanabe et al., 1988). Further, when

nutrient starvation. This may indicate that the toxicity is cells enter the late stationary growth phase as nutrients become so

not an accumulated biomolecule, but rather a physiological replete that energetically expensive processes like hemolytic

mechanism. scavenging are no longer worth the metabolic requirements,

There was no signiﬁcant difference between the mortality individual cells will yield metabolic activity and use stored energy

experienced by young versus mature brine shrimp. Furthermore, molecules to actively pursue nutrients and light elsewhere in the

the lack of signiﬁcant difference between the 24 and 48 h water column; in other words they increase motility (Smayda,

exposures suggests several characteristics of the toxic mechanism 1997b).

in H. akashiwo. First, the young brine shrimp are still physically The variability between H. akashiwo isolates observed should

attached to a nutrient-rich yolk that sustains cysts during serve as a cautionary note. The plasticity of toxic responses means

dormancy, thus the brine shrimp at this age are not actually that attempting to generalize the ecophysiology of H. akashiwo in

ingesting food particles from the external environment. Mature order to construct a more comprehensive framework of bloom

brine shrimp, however, have shed this yolk and will actively graze dynamics may be an impractical task. The behavior and toxicity of

on algae adjacent in the water column (Vanhaecke et al., 1981). individuals within a particular bloom will be highly dependent

Ingestion may not result in increased mortality, and thus the toxic upon the isolate(s) that dominate within the bloom. Subsequently,

compounds being produced by H. akashiwo may be acting research should focus on common regional H. akashiwo isolates to

externally on affected organisms (Honjo, 1993; Khan et al., develop an understanding at more local scales, and researchers

2008). Second, mortality did not signiﬁcantly increase after a should be hesitant to apply the lessons learned from a particular

second 24 h period (48 h total), supporting the hypothesis that any isolate to the bloom dynamics of another.

potential H. akashiwo toxins are highly labile and will not continue There was a signiﬁcant difference between ﬂoating and sinking

to affect other organisms for a notable period of time after initial fractions for H. akashiwo strains Can Ed and Spain, and although the

exposure (Grane´li and Hansen, 2006). Third, the struggling brine difference was not signiﬁcant for strain Can 764R, there was a clear

shrimp recovered after 72 h. These results contrast with HAB trend towards greater toxicity in sinking populations than in the

species that produce known toxic compounds, such as the ﬂoating or ascending population. This demonstrates that when

dinoﬂagellate Karenia mikimotoi, which has been shown to cause cellular toxicity is greatest, cells that tend to sink are likely going to

mortality for several days in bioassay experiments. be the most toxic individuals within a population during that

All H. akashiwo isolates were most toxic to A. salina at some speciﬁc timeframe.

point prior to the late stationary growth phase, and there was a As the population metabolizes surplus nutrients and moves into

clear trend towards greater toxicity under N-limited conditions. the late stationary growth phase, the most toxic cells will already be

At ﬁrst this seems counter-intuitive because it was previously at deeper depths in the water column. Nutrient deplete conditions

stated that: (i) cellular physiological stress is induced by nutrient (possibly coupled with the corresponding decrease in irradiance

limitation, (ii) increased stress likely increases the toxic proper- with depth) could drive these cells towards the benthic environment

ties of individual cells, and (iii) stress is highest in the late in search of new nutrients or induce vegetative resting cyst

stationary phase. Thus, it was originally hypothesized that formation (Yamochi, 1984). And as cells sink to nutrient-rich deeper

toxicity would likely be highest in the late stationary growth waters, cell toxicities may change with the altered physiological

phase as well. However, recent insights into the potential state. The potential effects of H. akashiwo on members of the benthic

mechanisms of toxicity in H. akashiwo suggest that toxicity community in marine coastal waters are not well understood, but it

may be an indirect byproduct of hemolytic iron scavenging (Ling is likely that benthic organisms are as susceptible to the toxicity of H.

and Trick, 2010). Iron is an essential cofactor in cellular enzymes akashiwo as pelagic organisms (Shikata et al., 2007). Cysts are

that regulate the uptake of nitrogen from the environment, and capable of rapid descent towards the benthos with sinking rates of

thus nitrogen limitation for phytoplankton communities may be about 5–11 m d , and thus it is these individuals that have the

the result of iron depletion (Utkilen and Gjolme, 1995). Hemolytic highest probability of acting as ‘‘seeds’’ during subsequent blooms

scavenging may be indicative of increased scavenging for peptides and seasons (Imai and Itakura, 1999).

L. Powers et al. / Harmful Algae 13 (2012) 95–104 103

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there may be a genetic component common to strains that are

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Within individual communities or blooms, if the individuals

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Grane´li, E., Johansson, N., 2003. Increase in the production of allelopathic sub-

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phase of individuals within a bloom. This study showed that

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in that individuals prone to sinking are also generally more toxic. If

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Lecourt, M.D., Muggli, D.L., Harrison, P.J., 1996. Comparison of growth and sinking

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thus subsequent blooms may be more conducive to becoming

toxic. Marshall, J.A., Ross, T., Pyecroft, S., Hallegraeff, G., 2005. Superoxide production by

marine microalgae. Mar. Biol. 147, 541–549.

Martinez, R., Orive, E., Laza-Martinez, A., Seoane, S., 2010. Growth response of six

Acknowledgement strains of Heterosigma akashiwo to varying temperature, salinity and irradiance

conditions. J. Plankton Res. 32, 529–538.

Morris, J.G., 1999. Harmful algal blooms: an emerging public health problem with

A NSERC Discovery Grant to C.G.T supports this work.[TS] possible health links to human stress in the environment. Annu. Rev. Energy

Environ. 24, 367–390.

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