Harmful Algae 60 (2016) 150–156

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Harmful Algae

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Selective growth promotion of bloom-forming raphidophyte

Heterosigma akashiwo by a marine bacterial strain

a a b a a,

Aiko Higashi , Yoshiko Fujitani , Natsuko Nakayama , Akio Tani , Shoko Ueki *

Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan

National Research and Development Agency, Japan Fisheries Research and Education Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan

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

Article history:

Received 17 June 2016 Algal bloom is typically caused by aberrant propagation of a single species, resulting in its predomination

Received in revised form 28 September 2016 in the local population. While environmental factors including temperature and eutrophication are

Accepted 14 November 2016 linked to bloom, the precise mechanism of its formation process is still obscure. Here, we isolated a

Available online 23 November 2016

bacterial strain that promotes growth of Heterosigma akashiwo, a Raphidophyceae that causes harmful

algal blooms. Based on 16S rRNA gene sequence, the strain was identiﬁed as Altererythrobacter

Keywords: ishigakiensis, a member of the class Alphaproteobacteria. When added to culture, this strain facilitated

Heterosigma akashiwo

growth of H. akashiwo and increased its cell culture yield signiﬁcantly. Importantly, this strain did not

Marine bacterium

affect the growth of other raphidophytes, Chattonella ovate and C. antiqua, indicating that it promotes

Selective growth promotion

growth of H. akashiwo in a species-speciﬁc manner. We also found that, in co-culture, H. akashiwo

Mutualism

suppressed the growth of C. ovate. When A. ishigakiensis was added to the mixed culture, H. akashiwo

growth was facilitated while C. ovate propagation was markedly suppressed, indicating that the presence

of the bacterium enhances the dominance of H. akashiwo over C. ovate. This is the ﬁrst example of

selective growth promotion of H. akashiwo by a marine bacterium, and may exemplify importance of

symbiotic bacterium on algal bloom forming process in general.

1. Introduction Short, 2012; Suttle, 2007; Wommack and Colwell, 2000), and

marine bacteria (Amin et al., 2012; Doucette et al., 1998; Mayali

Several species of unicellular algae are known to form blooms in and Azam, 2004) are known to play pivotal roles in algal

aquatic environments, thereby exerting a powerful inﬂuence on population control in nature. Particularly, studies on the inter-

their surrounding ecosystem. Harmful algal blooms (HAB) are actions between phytoplanktons and marine bacteria have

formed by noxious species and negatively impact the ecosystem, revealed both synergistic and parasitic interactions, resulting in

and are thus of particular interest in an environmental and propagation and fatality, respectively, of the algae (Amin et al.,

industrial context. Bloom status is determined by the speed of algal 2012; Buchan et al., 2014). Several reports have described the

propagation and disappearance. The propagation rate of bloom- existence of a characteristic marine bacterial assemblage associat-

forming algae has been shown to be affected by many environ- ed with a particular algal species (Barlaan et al., 2007; Burkholder

mental factors, including temperature, salinity, eutrophic condi- et al., 2007; Ferrier et al., 2002; Grossart et al., 2005). Furthermore,

tion, carbon dioxide concentration, and light intensity (Anderson one study showed that different algal species harbor different

et al., 2002; Backer and McGillicuddy, 2006; Eppley, 1972; bacterial assemblages with distinctive populations (Sapp et al.,

Hallegraeff, 1993; Heisler et al., 2008; Maso and Garces, 2006; 2007), implying that certain bacterial species preferably associate

Raven and Geider,1988; Smayda,1997; Smith and Schindler, 2009). with speciﬁc algal species. To improve our understanding of bloom

In addition, interactions with other organisms such as predator formation in nature, the implications of such interactions between

zooplanktons or other grazers (Demir et al., 2008; Graham and algae and bacteria should be investigated.

Strom, 2010; McManus et al., 2007; Tillmann, 2004; Xie et al., H. akashiwo is a noxious raphidophyte species that often forms

2008), viruses (Lang et al., 2009; Lawrence, 2008; Nagasaki, 2008; HAB during the summer, particularly in thalassic sea of the Paciﬁc-

rim area, including North and South America, Eastern Asia,

Oceania, and Northern Atlantic area (Black et al., 1991; Chang

et al., 1990; Honjo, 1993; Lackey and Lackey, 1963; Mackenzie,

* Corresponding author.

E-mail address: [email protected] (S. Ueki). 1991; O'Halloran et al., 2006; Park, 1991; Rensel et al., 1989; Rojas

http://dx.doi.org/10.1016/j.hal.2016.11.009

A. Higashi et al. / Harmful Algae 60 (2016) 150–156 151

de Mendiola, 1979; Taylor, 1993; Throndsen, 1969; Tseng et al., microorganisms in the treated culture. All sequenced amplicons

1993). While several reports have described marine bacteria that were identiﬁed as H. akashiwo chloroplast 16S rRNA, conﬁrming

kill H. akashiwo (Cho, 2012; Imai et al., 2001; Kim et al., 2007, that the cultures were successfully rendered bacteria-free by our

2009a, 1998, 2009b; Liu et al., 2008b; Lovejoy et al., 1998; Park method. The bacteria-free strains were maintained with the four

et al., 2010; Skerratt et al., 2002; Tarutani et al., 2000; Yoshinaga antibiotics thereafter.

et al., 1998), only one article has reported on a bacterial strain that

facilitates H. akashiwo propagation (Liu et al., 2008a). This strain 2.2. Isolation of Altererythrobacter ishigakiensis from H. akashiwo

facilitated the growth of a wide variety of algal species including strain H93616

raphidophytes, diatoms, cryptophytes, and chlorophytes by a

mechanism yet to be clariﬁed (Liu et al., 2008a). Nonaxenic H. akashiwo cultures (100 mL) containing 5 10

To gain more information about H. akashiwo-bacteria inter- cells were stab-pierced into soft-agar media containing 0.2% of

actions, we isolated bacteria associated with laboratory-main- bacto-agar and commercial Difco Marine Broth 2216. (Becton

tained H. akashiwo cultures. Here, we found that the bacterial Dickinson and Company, Franklin Lakes, NJ, USA). The bacteria

strain Altererythrobacter ishigakiensis YF1 facilitates H. akashiwo grown in the soft-agar media were streaked on Difco Marine

propagation, though it did not alter the growth rates of two other Broth 2216 solidiﬁed with 2% agar, and incubated at 25 C under

raphidophytes, Chattonella ovate and C. antiqua. This is the ﬁrst aerobic conditions. When colonies appeared, a partial sequence of

study that demonstrates selective raphidophyte growth facilita- the 16S rRNA gene was ampliﬁed using primers Eu8f and Eu1492r

tion by a marine bacterial strain. by colony PCR using a standard protocol (Devereux and Wilkinson,

2004), and its sequence was determined using Big Dye v3.1

2. Materials and methods (Applied Biosystems) according to the manufacturer’s instruction.

The sequences were used for identiﬁcation in the EZ-Taxon

2.1. Raphidophyte strains and media used for the experiments database (Chun et al., 2007; Kim et al., 2012).

H. akashiwo isolates, HaFk01 (isolated from Fukuoka Bay, 2.3. Analyzing effect of bacterial strains on growth of H. akashiwo and

Fukuoka prefecture, Japan), H93616 (isolated from Uranouchi Bay, Chattonella

Kochi Prefecture, Japan), HaTj01 (isolated from Tajiri Bay,

Hiroshima Prefecture, Japan), C. ovate CoTj23 (Tajiri Bay, Hiroshima The bacteria-free H. akashiwo and Chattonella strains were

Prefecture, Japan) and C. antiqua CaAg03 (Ago Bay, Mie Prefecture, transferred to an antibiotic-free growth medium and propagated

Japan) were used for this study. All strains used in this study were for three days at least twice to decrease the antibiotic concen-

originally established by isolating single algal cells from H. trations to <1/1000 of the treatment concentrations. During the

akashiwo or Chattonella bloom samples observed at the areas of preculture period, H. akashiwo was allowed to propagate at least 8

origin, then passaged in growth media without antibiotics. generations (in cell number, to >500 times) to acclimate to the

Modiﬁed SWM3 medium(Yamasaki et al., 2007) was used for growth condition without antibiotics. The bacterial strains were

algal growth medium, and the culture was maintained in an precultured in Marine Broth, and each 600-mL bacterial culture

environment-controlled chamber with a photoperiod (12 h of adjusted to OD600 = 0.01 (total 9 10 cells) was added to a 60-mL

2 1 $ Â

100 mmol mÀ sÀ light/12 h dark) at 25 C. algal culture. For controls, the same volume of Marine Broth was

The strains were rendered bacteria-free by being maintained in added to equivalent cultures. H. akashiwo, C. ovate, and C. antiqua

the growth medium supplemented with penicillin (100 units/mL), cells were innoculated at 100, 500 and 1000 cells/mL. These were

streptomycin (100 mg/mL), ampicillin (100 mg/mL), and kanamycin the minimum innoculation densities that allowed stable growth of

(60 mg/mL). To analyze whether any bacteria were present in the bacteria-free strains, thus adopted for the experiments.

algal cultures, 100 mL samples of raphidophyte cultures, each For cell enumeration, a MoxiZ Mini Automated Cell Counter (E.I.

5 6

containing 5 10 -10 /mL cells, were inoculated in commercial Spectra, LLC, Hailey, ID, USA) was used according to the

TM Â

Difco Marine Broth 2216 (Becton Dickinson and Company, manufacturer’s instructions. The algal species H. akashiwo and

Franklin Lakes, NJ, USA) solidiﬁed with 0.2% bacto-agar by piercing Chattonella are readily distinguishable on the counter because of

into the media to a depth of at least 5 cm using long pipette tips the difference in their diameters, which are 8–12 mm and

(Cappuccino and Natalie Sherman, 2013). The media were 18–30 mm, respectively; thus the two species could be enumerated

incubated at 25–28 C for two to three weeks, and conﬁrmed independently in mixed culture. The bacterial cell density was

the absence of microorganisms. We also conducted 16S rRNA gene enumerated by counting colony-forming units on solidiﬁed Marine

ampliﬁcation by PCR using primers Eu8f and Eu1492r (Devereux broth. Speciﬁc growth rate was calculated according to the

and Wilkinson, 2004), followed by cloning into pCR-Blunt II-TOPO equation k = ln(N2/N1)/(t2-t1), where N is concentration of cells

plasmid (Life Technologies, Carlsbad, CA, USA) and sequencing of and t is the date when the cell density was measured. All

the amplicon in ﬁfteen independent clones to detect traces of experiments were continued until the cell population reach to

Table 1

Bacterial strains isolated from H. akashiwo .

* $

Isolated strains Closest relatives Identities Accessions

(%)

YF1 Altererythrobacter ishigakiensis JPCCMB0017(T) 98.73 LC078994

AH2 Winogradskyella poriferorum UST030701-295(T) 100 LC078996

AH3 Stappia indica B106(T) 99.31 LC078997

AH4 Spongiibacterium ﬂavum A11(T) 97.31 LC078998

AH5 Alteromonas macleodii DSM 6062(T) 99.79 LC078999

The closest relatives were identiﬁed according to the identities of rRNA sequence.

The NCBI accession numbers.

152 A. Higashi et al. / Harmful Algae 60 (2016) 150–156

plateau, and terminated when the cell population started to decrease.

3. Results

3.1. Altererythrobacter ishigakiensis strain YF1 promotes

propagation of H. akashiwo

In the course of study about interaction between symbiotic

bacteria and H. akashiwo, we isolated ﬁve bacterial strains from

non-axenic cultures of H. akashiwo strains and identiﬁed them by

16S rRNA gene sequencing (Table 1). As a preliminary screening,

we tested the effect of all identiﬁed bacterial isolates on the

propagation of bacteria-free H93616 independently, and found

that one strain, identiﬁed as Altererythrobacter ishigakiensis strain

YF1, increased the maximum culture density of H93616, thus the

strain was subjected for further study. When YF1 was added to

bacteria-free cultures of the H. akashiwo strains HaFk01, H93616,

and HaTj01, the algal growth rate and the yield were increased, to

various degrees, in all tested isolates (Fig. 1A–C). As shown in

Table 2, addition of YF1 moderately promoted speciﬁc growth rate

during early stage of growth, while unchanged or suppresses

growth of the strains during mid to late log phase. The increases in

maximum culture densities were 29 1.2%, 41 1.4%, and

Æ Æ

71 3.0% for HaFk01, H93616, and HaTj01, respectively. These

data demonstrate that YF1 promoted growth speed of the algae at

early stage of the propagation and increase the maximum yield of

the culture. YF1, on the other hand, also propagated during the

growth phase of H. akashiwo (Fig. 1A–C), though it did not grow at

all when cultured alone in algal growth medium (Fig. 1A).

3.2. Growth of Chattonella was not altered by A. ishigakiensis strain

YF1

We next assessed the effect of YF1 on growth of two Chattonella

isolates, which are also bloom-forming Raphidophyceae. Two

bacteria-free isolates, C. ovate CoTj23 and C. antiqua CaAg03, were

cultured with or without YF1. Addition of YF1 did not affect the

growth of C. ovate CoTj23 (Fig. 2A) and C. antiqua CaAg03 (Fig. 2B).

YF1 did not signiﬁcantly affect propagation speed of either C. ovate

CoTj23 or C. antiqua CaAg03 (Table 3), and there was no signiﬁcant

difference between the maximum cell density of these Chattonella

strains with or without YF1. YF1 in the culture propagated to levels

comparable to those in H. akashiwo cultures (Fig. 1A–C), though

there was a long lag in the culture containing CaAg03.

3.3. A. ishigakiensis strain YF1 enhances predominance of H.

akashiwo over C. ovate

Fig. 1. Effect of the A. ishigakiensis strain YF1 on growth of three H. akashiwo strains

HaFk01 (A), H93616 (B), and HaTj01 (C). The growth of H. akashiwo bacteria-free

ﬁ ﬁ

To further con rm the species-speci c growth-promoting strains with (closed circles with solid lines) or without (open circles) YF1 was

effect of YF1 on H. akashiwo, we co-cultured H. akashiwo HaFk01 measured in cell number. Propagation of the bacteria in the culture (gray triangles)

or in algal growth medium without alga as a control (A, open triangles) was

and C. ovate CoTj23 with or without YF1 and evaluated the growth

measured in colony forming units. The data are presented as the mean standard

rates of the two algal species (Fig. 3). An addition of YF1 to the Æ

deviation (S. D.) of triplicate culture measured three times. Asterisks indicate

H. akashiwo

mixed culture promoted propagation while suppress- signiﬁcance of difference between H. akashiwo cell densities with or without YF1

ﬁ

ing C. ovate propagation in statistically signi cant manner (Fig. 3A). (Student’s t-test P values, P < 0.01). All experiments were repeated at least three

Speciﬁc growth rate of H. akashiwo was facilitated during early log times with similar results.

phase by addition of YF1, while that of C. ovate was markedly

suppressed (Table 4). Addition of YF1 increases maximum H. 4. Discussion

akashiwo density by 41% while suppresses Chattonella density to

62% at maximum (Fig. 3A). As a consequence, ratio of C. ovate to the To date, several bacterial strains have been reported to kill H.

total population decreased to 33 10.3–50 9.8% by addition of akashiwo, suggesting their important roles in bloom termination

Æ Æ

YF1 at different time points, in the co-culture experiment (Fig. 3B). (Cho, 2012; Imai et al., 2001; Kim et al., 2007, 2009a, 1998, 2009b;

These observations suggest, when in competing situations, that H. Liu et al., 2008a, 2008b; Lovejoy et al., 1998; Park et al., 2010;

akashiwo propagates more efﬁciently compared to C. ovate, and Skerratt et al., 2002; Tarutani et al., 2000; Yoshinaga et al., 1998).

YF1 enhances predominance of the former. Yet only one previous article has reported H. akashiwo growth

A. Higashi et al. / Harmful Algae 60 (2016) 150–156 153

Table 2 Table 3

* *

Speciﬁc growth rates of different H. akashiwo strains with or without A. Speciﬁc growth rates of Chattonella strains with or without A. ishigakiensis strain ishigakiensis strain YF1. YF1.

$ ^ $ $

Strains Stages Control + YF1 P Strains Stages Control + YF1

HaFk01 Early log 0.622 0.025 0.819 0.027 # CoTj23 Early log 0.413 0.098 0.406 0.048

Æ Æ Æ Æ

Mid-to-late log 0.795 0.024 0.677 0.016 # Mid-to-late log 0.363 0.004 0.383 0.020

Æ Æ Æ Æ

H93616 Early log 0.504 0.003 0.560 0.005 # CaAgo3 Early log 0.709 0.031 0.765 0.037

Æ Æ Æ Æ

Mid-to-late log 0.205 0.007 0.193 0.007 Mid-to-late log 0.258 0.013 0.237 0.021 Æ Æ Æ Æ

Speciﬁc growth rates calculated from the datasets presented in Fig. 2 are presented

HaTj01 Early log 0.509 0.012 0.533 0.010 #

Æ Æ as mean S. D. There was no signiﬁcance of difference between datasets obtained

Mid-to-late log 0.196 0.015 0.191 0.008 Æ

Æ Æ from the experiments with or without YF1.

* $

Speciﬁc growth rates calculated from the datasets presented in Fig. 1 are presented For CoTj23, early and mid-to-late log phase represent 0–5 dpi and 6–11 dpi,

as mean S. D. respectively, while for CaAgo3, 0–2 dpi and 3–6 dpi.

$ Æ

For all the strains, early and mid-to-late log phase represents 0–5 dpi and 6–10 dpi,

respectively.

ﬁ

^ culture, so the remaining bacteria grew to signi cant level in 2–3

Signiﬁcance of the differences, #, P < 0.01.

weeks, and became visible by the soft-agar assay. To minimize the

effect of bacteria contained in H. akashiwo culture during the

promotion by a marine bacterium (Liu et al., 2008a). Bacillus sp.

experiment, we adopted antibiotic treatment to render the algal

strain BBB25 promoted growth of all tested algal species, including

strains bacteria-free.

three raphidophytes, two diatoms and a chlorophyte. Thus, our

The strain YF1 was identiﬁed as A. ishigakiensis based on the

observation demonstrated selective growth promotion of H.

homology of 16S rRNA gene sequence. A. ishigakiensis strain

akashiwo by a bacterium strain for the ﬁrst time.

JPCCMB0017(T) was previously isolated from a marine sediment

To evaluate effect of bacterium on growth rate of H. akashiwo, it

collected from the coastal area of Okinawa Island, Japan

is important to utilize bacteria- free strains. To this end, we ﬁrst

tried to use H. akashiwo strains rendered bacteria-free by single cell

isolation. While this technique signiﬁcantly reduced bacteria in the

H. akashiwo culture, it does not eradicate bacteria contained in the

Fig. 3. (A) Effect of A. ishigakiensis strain YF1 on growth of H. akashiwo and C. ovate

in mix culture. H. akashiwo HaFk01 and C. ovate CoTj23 were inoculated, at 100 and

500 cells/mL, respectively, with or without YF1. The cell numbers of H. akashiwo

(circles with solid lines) and C. ovate (triangles with broken lines) cultured with

(closed symbols) or without (open symbols) YF1 were enumerated. The data are

presented as the mean S. D. of triplicate cultures measured three times. Asterisks

and ampersands indicate signiﬁcance of difference between H. akashiwo cell

densities and Chattonella cell densities, respectively, with or without YF1 (Student’s

t-test P values, P < 0.01). The experiments were repeated at least three times with

similar results.

(B) Effect of YF1 on ratio of C. ovate in the mix culture. The ratio of CoTj23 to total cell

Fig. 2. Effect of the A. ishigakiensis strain YF1 on growth of C. ovate CoTj23 (A) and C. numbers in mix culture at different time points were calculated based on the

antiqua CaAgo3 (B). Bacteria-free Chattonella strains with (closed circles with solid dataset presented in panel (A). The signiﬁcance of difference between the C. ovate

lines) or without (open circles) YF1 was measured in cell number. Propagation of ratios with (black columns) or without YF1 (white columns) at all three time points

the bacteria in the culture (gray triangles) was measured in colony forming units. were conﬁrmed by Student’s t-test as P value to be less than 0.01. Note that initial

The data are presented as the mean S. D. of triplicate culture measured three ratio of C. ovate to the total algal cells at the point of inoculation was 83.3% both in

times. All experiments were repeated at least three times with similar results. the presence or absence of YF1.

154 A. Higashi et al. / Harmful Algae 60 (2016) 150–156

Table 4

additional pathway through which H. akashiwo is allelopathic to

Speciﬁc growth rates of H. akashiwo and C. ovate in mixed culture with or without A.

Chattonella, resulting in more efﬁcient suppression of Chattonella ishigakiensis strain YF1. growth.

$ ^

Strains Stages Control + YF1 P While our results clearly demonstrated the mutualistic

HaFk01 Early log 0.841 0.018 0.912 0.010 # relationship between H. akashiwo and A. ishigakiensis strain YF1,

Æ Æ

Mid-to-late log 0.426 0.024 0.411 0.020

Æ Æ the mechanism underlying this relationship remained to be

clariﬁed. Several reports regarding diatoms have described

CoTj23 Early log 0.409 0.031 0.337 0.052 !

Æ Æ synergistic interactions between unicellular algae and marine

Mid-to-late log 0.687 0.062 0.654 0.078

Æ Æ

bacterium (Amin et al., 2012; Armbrust, 2009; Grossart, 1999).

Speciﬁc growth rates calculated from the datasets presented in Fig. 3 are presented

Microscale mucus region surrounding phytoplankton is termed

as mean S. D.

$ ‘phycosphere’, which is rich in organic matter secreted by the algal

For HaFk01, early and mid-to-late log phase represent 0–5 dpi and 6–11 dpi,

respectively, while for CoTj23, 0–2 dpi and 3–6 dpi. cells and provide nutritional source to habitat bacteria. In return,

ﬁ

Signi cance of the differences, #, P < 0.01; !, P < 0.05. bacteria in the phycosphere may provide some essential elements,

such as vitamins, to phytoplankton. For example, marine bacteria

(Matsumoto et al., 2011). This strain was characterized as a Gram- that synthesize B vitamins are known to support diatom

negative halophilic bacterium. Its colonies are orange-red due to its propagation in vitamin-limited conditions (Droop, 2007; Haines

high astaxanthin content, and it also accumulates other carote- and Guillard, 1974; Sañudo-Wilhelmy et al., 2006; Tang et al.,

noids including adonixanthin and zeaxanthin (Matsumoto et al., 2010). In our experiments, since B vitamins were supplemented in

2011). Like JPCCMB0017(T), YF1 expresses an orange-red color, the growth medium, addition of B vitamin-synthesizing bacteria to

suggesting that it also produces astaxanthin. In addition, unlike the culture is unlikely to affect algal growth. Also, nitrogen-ﬁxing

any other member of the genus Altererythrobacter, JPCCMB0017(T) marine bacteria convert nitrogen gas to forms that are more readily

was demonstrated to reduce nitrate (Matsumoto et al., 2011). biologically available to algae, such as ammonium (Foster et al.,

Bacteriochlorophyll a was absent from this strain. While strain YF1 2011). The previous study demonstrated that A. ishigakiensis strain

belongs to class Alphaproteobacteria, other bacteria that are JPCCMB0017(T), the closest relative of strain YF1, reduces nitrate

reported to possess algicidal or growth promoting effects belong (Matsumoto et al., 2011), and so YF1 may contribute to algal

to class Gammaproteobacteria, Bacilli, or Flavobacteira. Therefore, growth by modulating nitric compounds in the growth media.

ﬁ

there is no speci c preference in class of marine bacteria that These pathways are unlikely to be involved in the mode of

interact with H. akashiwo (Cho, 2012; Imai et al., 2001; Kim et al., interaction between YF1 and H. akashiwo, because if they were, the

2007, 2009a, 1998, 2009b; Liu et al., 2008a, 2008b; Lovejoy et al., growth of Chattonella should also be facilitated by the components

1998; Park et al., 2010; Skerratt et al., 2002; Tarutani et al., 2000; produced by the bacterium. Because YF1 did not promote

Yoshinaga et al., 1998). propagation of Chattonella, the synergistic interaction observed

YF1 promotes the growth of H. akashiwo, while the bacterium between H. akashiwo and YF1 may be independent of such

propagates only in the presence of the alga, demonstrating that the pathways.

bacterium and the alga are in a mutualistic relationship. This is also Alternatively, the bacterium could be involved in the metabo-

ﬁ

supported by the fact that the addition of ltrate of YF1 culture (i.e. lism of compounds produced by H. akashiwo that negatively

containing substance secreted from the strain but free from the live impact its growth. An example of this type of interaction has been

cells) to H. akashiwo did not have effect on growth of the alga (data reported for the Antarctic ice diatom Amphiprora kufferathii and its

not shown). Importantly, YF1 does not affect the growth of the two epiphytic bacteria (Hunken et al., 2008). In this case, the bacteria

Raphidophyceae members, C. ovate and C. antiqua, while YF1 detoxify hydrogen peroxide derived from photosynthesis by the

propagated in the presence of Chattonella sp., demonstrating that diatom, resulting in increased yield of the diatom. While YF1 is

YF1 and Chattonella sp. are in commensal relationship. These data thought to produce anti-oxidative compound astaxanthin (Mat-

indicate that, although its growth is supported by both Chattonella sumoto et al., 2011), and thus could have a similar detoxifying

sp. and H. akashiwo, YF1 only promotes the growth of H. akashiwo effect, but this is also unlikely since H. akashiwo and Chattonella are

in a selective manner. Moreover, in a mixed culture, we observed both photosynthetic and therefore produce reactive oxygen

intricate tripartite relationship among YF1, H. akashiwo and C. species as byproducts. If the detoxiﬁcation of H. akashiwo

ovate, based on competition between two algae, and selective metabolites by YF1 is involved in the effect, the substance

growth promotion of YF1 on H. akashiwo. When cultured as detoxiﬁed by YF1 may possess H. akashiwo-speciﬁc toxicity.

mixture, H. akashiwo outcompeted C. ovate in the absence of YF1. Additional modes of communication between phytoplanktons

The maximum cell number of each species in the mixed culture and bacteria through infochemicals have been reported (Amin

decreased compared to the single culture presumably because of et al., 2015; Hombeck and Boland, 1998; Pohnert and Boland, 1996,

competition over limited nutritional resources, while the extent of 2002). The modulation of such pathways by YF1, which stimulate

decrease was larger for C. ovate, showing that the H. akashiwo H. akashiwo selectively, may represent a potential mode of

propagates more robustly than C. ovate in the absence of YF1. H. interaction between H. akashiwo and YF1, and is now under

akashiwo may beneﬁt from more efﬁcient uptake of growth- investigation.

limiting nutritional factors compared to C. ovate. Altenatively, the Finally, studies to date demonstrated that H. akashiwo is

growth suppression of C. ovate may be conferred by allelopathy of mixotrophic. feeding on some marine bacteria (Jeong, 2011; Jeong

H. akashiwo. Such effect by H. akashiwo for C. antiqua (Qiu et al., et al., 2010; Seong et al., 2006). Importantly, C. ovate also exhibited

2011), Akashiwo sanguinea (Qiu et al., 2012), and Skeletonema feeding on some marine bacterium, while the extent of depen-

costatum (Yamasaki et al., 2007) are previously reported. dence on the bacteriovory is much smaller than H. akashiwo: the

Importantly, YF1 enhanced H. akashiwo growth selectively, while studies showed that H. akashiwo could acquire 12.5% of its body

the maximum Chattonella population was suppressed to a lower carbon from bacteria per day, while in case of C. ovata up to 1.4%.

density. These observations suggest that, in addition to selectively The difference in their bacteriovory activities may result in further

promoting the growth of H. akashiwo, YF1 enhances H. akashiwo difference in photosynthetic activities of two organisms, in this

dominance over Chattonella strains. This enhanced dominance may case, because YF1 is rich in astaxanthin (Matsumoto et al., 2011),

further strengthen these H. akashiwo’s antagonistic effect on which may detoxify reactive oxygen species as photosynthesis

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