Science of the Total Environment 926 (2024) 171688

Available online 15 March 2024

0048-9697/© 2024 Elsevier B.V. All rights reserved.

A laboratory study of the increasing competitiveness of Karenia mikimotoi

under rising CO2 scenario

Chao Wang 1

, Renjun Wang *,1

, Lingna Meng , Wenjing Chang , Junfeng Chen , Chunchen Liu ,

Yuhao Song , Ning Ding , Peike Gao *

College of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, PR China

HIGHLIGHTS GRAPHICAL ABSTRACT

• Rising CO2 level promoted the growth of

Karenia mikimotoi.

• Rising CO2 weakened allelopathic effects of Ulva pertusa on K. mikimotoi.

• High CO2 level disturbed the synthesis

of free fatty acids in U. pertusa,

decreasing its allelopathic effects.

• Rising CO2 increases the outbreak risk of

K. mikimotoi.

ARTICLE INFO

Editor: Julian Blasco

Keywords:

Ocean acidification

Allelopathy

Karenia mikimotoi

Ulva pertusa

Metabolomics

ABSTRACT

Ocean acidification (OA) driven by elevated carbon dioxide (CO2) levels is expected to disturb marine ecological

processes, including the formation and control of harmful algal blooms (HABs). In this study, the effects of rising

CO2 on the allelopathic effects of macroalgae Ulva pertusa to a toxic dinoflagellate Karenia mikimotoi were

investigated. It was found that high level of CO2 (1000 ppmv) promoted the competitive growth of K. mikimotoi

compared to the group of present ambient CO2 level (420ppmv), with the number of algal cell increased from

32.2 × 104 cells/mL to 36.75 × 104 cells/mL after 96 h mono-culture. Additionally, rising CO2 level weakened

allelopathic effects of U. pertusa on K. mikimotoi, as demonstrated by the decreased inhibition rate (50.6 % under

the original condition VS 34.3 % under the acidified condition after 96 h co-culture) and the decreased reactive

oxygen species (ROS) level, malondialdehyde (MDA) content, antioxidant enzymes activity (superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT)

and non-enzymatic antioxidants (glutathione (GSH) and ascorbic acid (ascorbate, vitamin C). Indicators for cell

apoptosis of K. mikimotoi including decreased caspase-3 and -9 protease activity were observed when the cocultured systems were under rising CO2 exposure. Furthermore, high CO2 level disturbed fatty acid synthesis

in U. pertusa and significantly decreased the contents of fatty acids with allelopathy, resulting in the allelopathy

weakening of U. pertusa. Collectively, rising CO2 level promoted the growth of K. mikimotoi and weakened

* Corresponding authors.

E-mail addresses: wangrenjun2002@126.com (R. Wang), gpkyll-001@163.com (P. Gao). 1 Chao Wang and Renjun Wang contributed equally to this work.

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

https://doi.org/10.1016/j.scitotenv.2024.171688

Received 2 September 2023; Received in revised form 7 December 2023; Accepted 11 March 2024

Science of the Total Environment 926 (2024) 171688

2

allelopathic effects of U. pertusa on K. mikimotoi, indicating the increased difficulties in controlling K. mikimotoi

using macroalgae in the future.

1. Introduction

Climate change induced by anthropogenic activities drives the

occurrence of warming and ocean acidification (OA) of the surface

layers. These changes affect the composition of phytoplankton communities and modify the distribution and intensity of multiple costressors (Griffith and Gobler, 2020; Seto et al., 2019), which, in turn,

pose great challenges to water space ecosystems. The oceans in worldwide have absorbed nearly 30 % of the total carbon emissions since the

industrial revolution, which cause pCO2 increase from roughly 280

ppmv to 420 ppmv today, making ocean pH decrease by 0.1 (Dupont and

Portner, ¨ 2013). The recent prediction indicates the ocean pH levels will

increase by 0.3–0.4 by 2100 and 2300 with the present resource utilization structure followed (Zeebe et al., 2008). Generally, OA causes

survival pressure among sensitive marine species, which has potential

impacts on the physiological and ecological features of phytoplankton

(Brandenburg et al., 2019; Van de Waal et al., 2013).

Harmful algal blooms (HABs) species are of great concern as the

negative effects they cause among the many phytoplankton species that

will be affected by OA. Considering the impact of climate change, it is

increasingly recognized that OA plays an important role in the intensification of several HABs (Glibert, 2020; Gobler et al., 2017; McKibben

et al., 2017). For instance, an increase in surface water temperature may

enhance HABs species growth rates and result in a geographic redistribution of HABs (Chateau-Degat et al., 2005; Ramilo et al., 2021; Tester,

1994). Moreover, CO2-concentrating mechanisms (CCMs), a survival

strategy to increase the CO2 concentration in the proximity of Rubisco

enzymes, has been evolved as the low diffusion rate of CO2 in the marine

environment (Coleman, 2010; Reinfelder, 2011). Compared to other

phytoplankton, the metabolic costs for CCMs are expected to downregulate under elevated pCO2 as the Rubisco in dinoflagellates has a

lower affinity for CO2, which cause dinoflagellates may benefit from the

rising CO2 levels (Eberlein et al., 2014; Van de Waal et al., 2019). Karenia mikimotoi, a HAB-forming dinoflagellate species, attracts much

attention due to the environmental and economic problems it caused (Li

et al., 2017; Li et al., 2019). It has been evidenced that hemolytic toxins

and cytotoxins are the main causes of marine organism mortality, which

disturbs the marine ecosystem (Neely and Campbell, 2006; Satake et al.,

2002). More importantly, the competitive advantage of K. mikimotoi is

expected to increase in the context of OA, which may increase the

severity of HABs (Zhang et al., 2022).

Utilization of allelochemicals secreted from macroalgae to inhibit

microalgal overgrowth has become one of most effective approaches for

controlling K. mikimotoi. Some natural products, such as 1-O-palmitoyl3-O-β-D-galactopyranosyl glycerol, 1-β-D-ribofuranosyluracil and 3-

hydroxymethyl-pyrrolopiperazine-2,5-dione secreted by Ulva prolifera

and Gracilaria lemaneiformis, have been identified and show great inhibition activities to K. mikimotoi (Sun et al., 2019; Sun et al., 2021). There

is, however, an abundance of evidence that metabolic selection and

physiological indictors of macroalgae, such as U. prolifera and

G. lemaneiformis, are affected to some extent with an increasing OA.

Besides, costs of CCMs are reduced with an increasing pCO2, which in

turn regulates other metabolite processes to accommodate to the

changing marine environment, especially changes in growth, calcification rate and pigment composition (Gao et al., 2016; Koch et al., 2013;

Yang et al., 2020; Zou and Gao, 2014). Ulva linza has shown inhibitory

effects on the growth of Skeletonema costatum, while acidification

treatment decreases its allelopathy at simulated future CO2 (1000 ppm)

level (Gao et al., 2019). Moreover, an increase in pCO2 affects the production and release of various secondary metabolites, which alleviates

their allelopathy to HABs species. For example, contents of phenolic

compounds in multitude macro brown algae are decreased under high

CO2 concentration exposure, which reduces the allelopathy on red tide

microalgae (Betancor et al., 2014; Tan et al., 2019). However, few

studies have systematically analysed the allelopathy of macroalgae on

K. mikimotoi under increasing CO2 levels.

Meng et al. investigated that acidified (pH = 7.8) seawater reduced

the allelopathy of Ulva pertusa on K. mikimotoi (Wang et al., 2023a), and

increased cell density of K. mikimotoi driven by excessive CO2 stimulation may be the main reason for weakening its allelopathy, which was

verified at the transcriptional level to some extent (Zhang et al., 2022).

However, our knowledge on the response mechanism of K. mikimotoi to

U. pertusa and the alteration of allelochemicals secreted by U. pertusa

under OA conditions remains limited. Thus, a series of culture experiments were carried out to explore response of K. mikimotoi with various

U. pertusa concentrations (0, 2.5, 5, and 10 gFW/L) co-cultured and

changes of allelochemicals secreted by U. pertusa under original (420

ppmv) and acidified (1000 ppmv) seawater conditions, which aimed to

access the possibilities to prevent HABs using macroalgae under future

seawater conditions.

2. Material and methods

2.1. Culture conditions and experimental design

K. mikimotoi cultures were provided by Ocean University of China,

and was cultured in f/2 medium in an illumination CO2 incubator at 25

± 2 ◦C (RX-400C-CO2, Changzhou Haibo Equipment Company Limited),

with photon flux density of 54 μmol photon m− 2 s

− 1 and a light-dark

cycle of 12:12 h. U. pertusa was obtained from Jinsha Beach Park,

Yantai, China, and was domesticated in same cultivation conditions of

K. mikimotoi after the surface of algae was cleaned with sterilized

seawater. K. mikimotoi at exponential growth phase were inoculated into

200 mL of media with an inoculation density of 8 × 104 cells/mL, and

U. pertusa was added into the media with the concentration of 0, 2.5, 5,

and 10 gFW/L, respectively, which were carried out in triplicate. Besides, two CO2 levels, 420 ppmv (pH 8.2; current ambient CO2 level) and

1000 ppmv (pH 7.8; predicted CO2 concentration by 2100), were evaluated and the CO2 concentrations were achieved by gentle bubbling

with 0.22 μm filtered ambient air and air/CO2 mixtures with a variation

of <5 %.

2.2. Population densities and soluble proteins analysis

The number of K. mikimotoi cells at 0, 24, 48, 72, 96 h was measured

using an optical microscope. A bradford protein assay kit ((Beyotime

Institute of Biotechnology, Shanghai, China) was used for analysing

changes of soluble proteins in K. mikimotoi under various treatments.

Briefly, 40 mL 72 h-old algal culture was centrifuged at 4 ◦C for 15 min

at 1000 ×g to collect algal cells, and the cells were washed twice using

phosphate-buffered saline (PBS), which were broken using an ultrasonic

cell disruption system (JY92-2D, NingBo Scientiz Biotechnological Co.,

China) at 200 W for 5 min below 4 ◦C (ultrasonic time, 5 s, rest time, 10

s). Changes of soluble proteins were analysed according to the kits’ instructions using above supernatant after centrifugation (4 ◦C, 1000 ×g,

15 min).

2.3. Alteration of reactive oxygen species of K. mikimotoi

Algal cells typically produce reactive oxygen species (ROS) under

abiotic stresses and the change of ROS content reflects the oxidative

damage of algae to some extent (Rezayian et al., 2019). A ROS assay kit

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(Beyotime Institute of Biotechnology, Shanghai, China) was used to

detect intracellular ROS based on the degree of non-fluorescent DCFHDA (2′,7′-dichlorodihydrofluorescein diacetate acetyl ester). In brief,

cells of K. mikimotoi were collected via centrifugation at 4 ◦C for 15 min

at 1000 g after 24 h, 48 h and 72 h cultivation. The supernatants were

discarded, and 1 mL DCFH-DA was added to the algal cells with incubation treatment at 37 ◦C in darkness for 30 min. The fluorescence intensity was detected by a flow cytometer (Novocyte 2040R, ACEA, USA)

with excitation wavelength of 488 nm and emission wavelength of 525

nm, to represent ROS production (Wang et al., 2023b).

2.4. Oxidative stress assay

Excessive production of ROS induces lipid peroxidation damage and

antioxidative defense system has been evolved to cope on the oxidative

damage of ROS in algae (Bhattacharya et al., 2010; Tziveleka et al.,

2021). Antioxidant enzymes and non-enzymatic antioxidants are the

primary defensive strategies to decrease the damage of ROS to algal

cells, which comprises superoxide dismutase (SOD), catalase (CAT),

glutathione peroxidase (GPX), glutathione reductase (GR), peroxidase

(POD), glutathione (GSH) and ascorbic acid (ascorbate, vitamin C) (Gill

and Tuteja, 2010). Maondialdehyde (MDA), a secondary lipid peroxidation product, is usually regarded as an identifier for oxidative hurt

(Valavanidis et al., 2006). The activities of SOD, CAT, GPX, GR and POD,

and the contents of GSH, vitamin C and MDA in various treatments were

measured after 72 h cultivation. Firstly, 40 mL of K. mikimotoi cells were

collected by centrifugation at 1000 g at 4 ◦C for 15 min. Then, the

separated algal cells were resuspended twice by PBS and ultrasonically

smashed following above crushing conditions in Section 2.2. Finally,

related detection indexes in the algae were measured using a Synergy H1

microporous reader (BioTek. America) following the respective assay

kits (Beyotime Institute of Biotechnology, Shanghai, China).

2.5. Analysis of cell apoptosis

The cell apoptosis rate of K. mikimotoi under the stress of U. pertusa

with conditions of natural and acidified seawater was detected using an

annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology of Shanghai, China) by flow cytometry (FCM). Briefly,

K. mikimotoi cells in each treatment with 72 h cultivation were harvested

and suspended with PBS. Annexin V-FITC and PI were added to stain

algal cells after collecting the algal cells, and the suspension was incubated at 25 ◦C for 20 min, which was monitored by the FL-1 channel and

FL-2 channel in the FC 500 MPL flow cytometer (Novocyte2040R, ACEA,

USA), respectively (Wang and Liu, 2022).

The caspase-cascade system plays an important role in regulating cell

apoptosis, and two types of caspases, initiator caspases and executioner

caspases, are essential in this system, which includes caspase-9, caspase3 and so on (Al Monla et al., 2020; Kim and Kim, 2018). Caspase-3 and

caspase-9 protease activity detection kit (Beyotime Institute of

Biotechnology of Shanghai, China) were used to detect the activity of

caspase-9 and caspase-3 protease in K. mikimotoi with 72 h cultivation,

and its fluorescence intensity was monitored Synergy H1 microporous

reader (BioTek Instruments, Inc., America) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

2.6. Alteration of algal inhibition mode of U. pertusa under various pCO2

levels exposure

To explore the alternation of algal inhibition mode and allelochemicals secreted by U. pertusa to acidified seawater, separated coculture

experiment, assay of filtered water from U. pertusa culture and detection

of free fatty acid of U. pertusa were carried out.

Separated coculture experiment: U. pertusa with concentration of 10

gFW/L was cultured in f/2 medium for 3 days under the normal and

acidified conditions, and filtrate of above culture system was obtained

using filter with 0.2 μm pore size to explore the growth inhibition rate at

0, 24, 48, 72 and 96 h.

Assay of filtered water from U. pertusa culture: U. pertusa and

K. mikimotoi were separated by a piece of bolting-silk (1 μm), which

allowed allelochemicals of U. pertusa to pass through. Above systems

were cultured in the normal and acidified conditions, and the growth

inhibition rate were monitored at 0, 24, 48, 72 and 96 h.

Detection of free fatty acid of U. pertusa: U. pertusa with concentration of 30 gFW/L was cultured in f/2 medium for 3 days under the

normal and acidified conditions. 10 g fresh U. pertusa were washed with

sterile seawater and grinded for further extraction. Metabolites extraction, qualitative and quantitative analysis and data pre-processing (Kuhl

et al., 2012) were outsourced at Biotree Biomedical Technology Co., Ltd.

(Shanghai, China). Algal samples were analysed using gas chromatography–mass spectrometry (GC–MS) analysis in the following steps:

(1) The system utilized a DB-FastFAME capillary column (90 m ×

250 μm × 0.25 μm) and helium was used as the carrier gas with the front

inlet purge flow and column pressure set as 3 mL min− 1 and 46 psi.

(2) 1 μL aliquot of the injected in split mode (5:1), and the programmed temperature steps were as follows:

The initial temperature was kept at 75 ◦C (hold on 1 min); raised to

200 ◦C at a rate of 50 ◦C min− 1 (hold on 15 min); raised to 210 ◦C at a

rate of 2 ◦C min− 1

(hold on 1 min); raised to 230 ◦C at a rate of 10 ◦C

min− 1

(hold on 16.5 min). The injection, transfer line, quad and ion

source temperatures were 240 ◦C, 240 ◦C, 230 ◦C and 150 ◦C,

respectively.

(3) The energy was − 70 eV in electron impact mode. Moreover, the

mass spectrometry data were acquired in Scan/SIM mode with the m/z

range of 33–400 after a solvent delay of 7 min.

2.7. Statistical analysis

All experiments were performed in triplicate, and the results were

expressed as the mean ± standard deviation (SD). Differences between

various groups were analysed by one-way ANOVA and t-text in GraphPad Prism 8. Values of P < 0.05 were considered statistically significant.

3. Results

3.1. Effects of U. pertusa on population densities and contents of soluble

proteins in K. mikimotoi cells under original and acidified conditions

Alteration of algal density of K. mikimotoi in various treatments was

shown in Fig. 1A. High U. pertusa concentrations (≥ 2.5 gFW/L) significantly inhibited algal growth, and inhibitory effect increased with

increasing concentration of U. pertusa, with the inhibitory rates ranging

from 16.8 % to 50.6 % after 96 h co-culture. Moreover, long period

seawater acidification exposure was beneficial for the growth of

K. mikimotoi, and the inhibitory effect of U. pertusa was alleviated to

some extent, with the inhibitory rate ranging from 4.9 % to 34.3 % after

96 h co-culture.

Algal cell soluble proteins are consist of various enzymes involved in

metabolic progress, which act as important indicator to different stress

factors (Bajguz and Piotrowska-Niczyporuk, 2014). It was observed that

high U. pertusa concentrations significantly decreased soluble protein

contents, and a dose-dependent effect was shown in Fig. 1B. Compared

with control, the soluble protein contents were down-regulated under

acidification treatment. In addition, soluble protein contents in acidification groups with various concentrations U. pertusa co-cultured were

up-regulated compared with those in normal group, which illustrated

that OA lowered the allelopathy effect of U. pertusa on K. mikimotoi.

3.2. Effects of U. pertusa on oxidative stress and antioxidant system in

K. mikimotoi cells under original and acidified conditions

ROS, a secondary messengers in algae, usually responses to

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environmental stresses (Rezayian et al., 2019). The ROS levels of algae

cells in co-culture groups were significantly increased under the original

and acidified conditions after 24 h and 48 h exposure, and the changing

trend showed a dose-dependent trend while a slight decrease of all

groups was shown at 72 h. Moreover, the increases in pCO2 significantly

promoted the production of ROS in K. mikimotoi under the mono-culture

group, which meant the acidified conditions induced the oxidative stress

of algae cells. However, acidification treatments attenuated the production of ROS when the algal cells were under same concentration of

U. pertusa exposure. The relative contents of ROS were 142 %, 159 %

and 169 % in 2.5 g/L, 5 g/L and 10 g/L U. pertusa treatment group

compared to the control group, while those ROS contents changed to

130 %, 156 % and 153 % under higher CO2 level exposure, respectively

(Fig. 2A). This suggested that U. pertusa exposure and acidification

treatments both induced overproduction of, while OA alleviated the ROS

production driven by allelopathic effects of U. pertusa.

The overproduction of ROS induced the lipid peroxidation The produced MDA increased significantly in different co-cultured groups

compared with the control group under the original and acidified conditions. The MDA contents of the control group, 2.5 g/L, 5 g/L and 10 g/

L U. pertusa treatment groups were 0.0927, 0.1529, 0.2541 and 0.3642

μmol/L under the original condition, respectively, while above contents

changed into 0.1215, 0.1346, 0.2387 and 0.3423 μmol/L under the

acidified condition. Moreover, acidification treatment increased the

MDA contents in control groups, and the MDA contents in algal cells

were significantly decreased in acidification treatments compared with

normal groups with same U. pertusa concentration exposure. The MDA

contents in algae cells were 3.93 times higher than the control group

under the original group, which was much higher than that of 2.82 times

under acidified treatment (Fig. 2B).

Algal cells have antioxidant enzymes system and antioxidant metabolites to defense produced ROS (Pikula et al., 2021). Fig. 3A - E

showed that antioxidant enzymes in algal cells, including SOD, POD,

GPx, GR and CAT, were influenced by U. pertusa exposure and acidified

treatment. Compared to the control group, the activity of SOD, POD,

GPx and GR in algae cells significantly increased with U. pertusa cocultured in both original and acidified treatment, and the upward

trend was positively correlated with the concentration of U. pertusa.

Moreover, magnitude of changes in some antioxidant enzymes activity

were decreased compared with control groups when the co-cultured

systems were under acidified treatment. The SOD POD and GR activity

of algae cells in groups with 2.5, 5, 10 g/L U. pertusa co-cultured were

0.09, 0.28,1.97, 2.35, 4, 4.8, 1.27, 1.81, 2.43 times higher than that in

the control group under the original condition, while acidification

treatment made them decrease to 0.06, 0.18, 1.51, 1.57, 2.45, 3.29,

1.35, 1.66 and 1.83 times, respectively (Fig. 3A. B. D). In Fig. 3C, the

GPx activity of algae cells in co-cultured systems with 2.5, 5, 10 g/L

U. pertusa were 1.31, 1.55 and 1.71 times higher than that in the control

group under the original condition, which decreased to 1.18, 1.77 and

2.03 times under the condition of seawater acidification, respectively. In

addition, change trend of CAT activity was different from that of other

antioxidant enzymes, whose change trend was increased firstly and

Fig. 1. Alternation of algal density (A) and soluble protein contents (B) in K. mikimotoi with various U. pertusa concentrations co-cultured when exposed to different

pCO2 levels.

Fig. 2. Alternation of the ROS levels (A) and MDA (B) contents in K. mikimotoi with various U. pertusa concentrations co-cultured when exposed to different

pCO2 levels.

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decreased then with increasing U. pertusa concentrations (Fig. 3E).

Changes in contents of antioxidant substances, glutathione (GSH)

and ascorbic acid (Vc) were shown in Fig. 4. U. pertusa significantly

induced the production of GSH and Vc under the original and acidified

conditions, and the metabolites contents showed a dose-dependent

trend. Moreover, acidification treatment significantly induced the production of GSH and Vc in K. mikimotoi under the mono-culture group and

reduced changes in contents of GSH and Vc caused by allelopathy of

U. pertusa. The contents of GSH and Vc of algae cells in groups with 2.5,

5, 10 g/L U. pertusa co-cultured were 1.78, 2.83, 3.82, 1.37, 1.77 and 2.4

times higher than that in the control group under the original condition,

which decreased to 1.55, 2.52, 3, 1.37, 1.65 and 2 times under acidification treatment.

3.3. Effects of U. pertusa on cell apoptosis in K. mikimotoi cells under

original and acidified conditions

The effects of U. pertusa exposure and acidification treatment on the

induction of apoptosis were performed by flow cytometry using

annexinV-FITC/PI double staining. We observed dose-dependent effects

of U. pertusa under original condition, i.e., an increasing rate of early and

late apoptotic cells with co-cultured concentrations of U. pertusa

increased compared to the control. In 10 gFW/L U. pertusa co-cultured

group, percentage of early apoptotic cells increased from 12.6 % to

33.72 %, and that of late apoptotic cells increased from 2.51 % to 15.21

% when co-cultured systems were under original condition, while above

percentage increased from 13.06 % and 6.79 % to 30.53 % and 16.34 %

Fig. 3. Alternation of the SOD (A), POD (B), GPx (C), GR (D) and CAT (E) activity in K. mikimotoi with various U. pertusa concentrations co-cultured when exposed to

different pCO2 levels.

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under the acidified condition, respectively. Moreover, acidification

treatment promoted apoptosis of K. mikimotoi in the mono-culture

group, and percentage of early and late apoptotic cells increased from

12.6 % and 2.51 % to 13.06 % and 6.79 %, respectively. In addition,

acidification treatment weakened the allelopathy of U. pertusa, and the

percentage of cell apoptosis under original condition was higher than

that in group with acidification treatment (Fig. 5), which could be

obvious in Fig. 6C.

Fig. 4. Changes of the GSH (A) and Vc (B) contents in K. mikimotoi with various U. pertusa concentrations co-cultured when exposed to different pCO2 levels.

( ) ( ) ( )

( ) ( )

( )

( )

( )

Fig. 5. Flow cytometry analysis revealed the effects of U. pertusa and acidification treatment on cell apoptosis of K. mikimotoi after 72 h treatment.

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Caspase-3 and -9 are key caspases involved in apoptosis, and changes

of caspases contents reveal the degree of cells apoptosis. It was obvious

that acidification treatment significantly drove the elevation of caspase3 and -9 protease activity in mono-culture system for 72 h. Moreover, a

dose-dependent effect was shown in Fig. 6A and B with increasing

U. pertusa exposure concentrations, while caspases activity in algae with

U. pertusa co-cultured were significantly inhibited under acidified condition, which illustrated acidification treatment relieved the allelopathy

of U. pertusa.

3.4. Responses of fatty acids secreted by U. pertusa to ocean acidification

Fig. 7 showed the K. mikimotoi inhibition rate of allelochemicals in

filtered water and separated coculture. Acidification treatment significantly alleviated inhibitory effect of U. pertusa. After 96 h exposure, the

inhibition rate reached to 43.32 % and 71.85 % in filtered water and

separated coculture assays when above systems were under the normal

condition, while above inhibition rate decreased to 33.22 % and 63.18

% under the acidified condition, respectively. Furthermore, fatty acids

in U. pertusa were specially analyzed as they are one of the most

important categories with allelopathy effect. Therefore, an assay of fatty

acids targeted detection was performed to investigate the effect of

Fig. 6. Alternation of caspases contents and cell apoptosis rate of K. mikimotoi with various U. pertusa concentrations co-cultured under the normal and acidified

conditions. (A) Changes of caspase-3 protease activity. (B) Changes of caspase-9 protease activity. (C) Apoptosis rate of K. mikimotoi under different treatments.

Fig. 7. Changes in effects of allelochemicals secreted by U. pertusa under the normal and acidified conditions. (A) Assay of filtered water from U. pertusa culture. (B)

Inhibition rate of allelochemicals to K. mikimotoi in separated coculture experiment.

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acidification treatment on fatty acid species and contents.

It was obvious that 30 free fatty acids were detected, which included

20 unsaturated fatty acids and 10 saturated fatty acids, 12 and 18 metabolites were down- and up- regulated when U. pertusa was under

acidification treatment (Fig. 8A). A total of 14 free fatty acids were hit in

fatty acid biosynthesis and biosynthesis of unsaturated fatty acids,

which included some metabolites with allelopathy, such as linoleic acid,

arachidic acid, stearic acid, palmitic acid, adrenic acid, myristic acid,

gamma- linolenic acid and arachidonic acid (Fig. 8B). Although above

fatty acids were down- and up-regulated to various degrees, contents of

them presented large differences. Contents of palmitic acid reached to

142.24 μg/g and 136.474 μg/g in NC and AC, while contents of

Fig. 8. Alteration of free fatty acid in 10 g fresh U. pertusa under normal condition (NC) and acidified condition (AC). (A) Heatmap of free fatty acid in NC and AC. (B)

Related metabolic pathways of detected free fatty acid in U. pertusa (red and black metabolites were up- and down-regulated free fatty acids, respectively). Black

metabolites with the dotted line were not detected. Processes with solid lines are one-step reactions, while those with dotted lines are more than one-step reactions.

(C) and (D) Alteration of free fatty acid contents in NC and AC, which were hit in related metabolic pathways.

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arachidonic acid, the highest level of upregulated metabolite, only

reached to 12.65 and 14.63 μg/g, respectively (Fig. 8C. D). Moreover,

contents of the left free fatty acids were shown in Fig. S1, which included

3 and 1 free fatty acids with allelopathy were down- and up-regulated.

Overall, contents of downregulated free fatty acids reached to 420.1

and 396.655 μg/g when U. pertusa were under normal and acidified

conditions, while up-regulated metabolites reached to 92.04 and 97.63

μg/g. Overall, acidification treatment greatly disturbed the synthesis

and release of fatty acids, which may been the main causes of reduced

allelopathic effects of U.

pertusa.

In summary, these results can be integrated to form a model. On the

one hand, rising CO2 level promoted the growth of K. mikimotoi, on the

other hand, algal density of K. mikimotoi significantly decreased with

various U. pertusa co-cultured, while acidification treatment weakened

allelopathic effects of U. pertusa on K. mikimotoi, which was due to the

decreased free fatty acid with allelopathy of U. pertusa under acidification conditions. Though both the acidification treatments and co-culture

treatments induced the overproduction of ROS in K. mikimotoi, the

acidification stress seemed to reduce the production of ROS in

K. mikimotoi induced by U. pertusa. In contrast to above alternative ROS

levels, several antioxidant enzymes and substances, including SOD,

POD, GPx, GR, CAT, GSH and Vc, presented the opposite changes under

acidified conditions to eliminate ROS. Above changes directly decreased

apoptosis rate of K. mikimotoi, making more algal cells survive under

future seawater conditions. All in all, acidification treatment reduces the

synthesis of allelochemicals of U. pertusa while promoting the growth of

K. mikimotoi, which collectively promoted an apparent increase of

K. mikimotoi.

4. Discussion

In the study, the suppression effect showed a dose-dependent trend

with increased U. pertusa exposure concentrations, and acidification

treatment (1000 ppmv) groups exhibited higher K. mikimotoi cell density,

suggesting excess CO2 promoted K. mikimotoi growth and relieved the

allelopathy of U. pertusa. High CO2 level lowered oxidation damage of

U. pertusa to K. mikimotoi and promoted responses of antioxidant system,

which greatly alleviated cell apoptosis of K. mikimotoi. Furthermore,

acidified treatment changed effects of allelochemicals in U. pertusa, as

demonstrated by the alteration of free fatty acids in U. pertusa. Collectively, our results indicated that elevated CO2 level increased outbreak

risks of K. mikimotoi due to the benefits of OA to K. mikimotoi cell density

and the decrease of allelopathic effect of U. pertusa.

The rising CO2 levels in seawater was favourable to the growth of

some HABs species, which was attributed to the regulation of CCMs. These

carbon-concentrating mechanisms may be active with an increasing CO2

level to reduce the energy cost of accessing carbon and the energy saved

largely stimulates algal growth and metabolism(Wu et al., 2014). In fact,

a study demonstrated that cell density of K. mikimotoi was inhibited

when exposed to short period of seawater acidification and caspase-3

and -9 protease activity were stimulated, which induced the increase

of apoptotic rate. However, the growth of K. mikimotoi was stimulated to

some extent under prolonged stressing time, which was similar to the

responses of K. mikimotoi to seawater acidification with time went in this

study (Li et al., 2021b).

ROS, acts as signalling molecules in algae, drives cellular responses

to OA as well (Mullineaux et al., 2018). It has been reported that OA

stress induced the accumulation of ROS and results in oxidative damage

(Liu et al., 2021), and similar results were observed in our study.

Enzymatic and non-enzymatic antioxidant systems are involved in

maintaining redox homeostasis in algal cells. Antioxidant enzyme,

including SOD, POD, CAT and so on, are essential to catalyse some

reactive oxygen molecules such as O2− and H2O2 (Wang et al., 2021). In

addition, GPx and GR play an important role in blocking further damage

to algal cells by reactive oxygen species (Margis et al., 2008). The

present study revealed that SOD, POD and CAT activities were significantly increased after OA exposure for 72 h, similar to the responses of

Chlorella vulgaris to OA stress (Xia et al., 2018). In addition, Nonenzymatic antioxidants such as GSH and Vc also play an important

role in removing ROS (Wu et al., 2021). In this study, the synthesis of

GSH and Vc were promoted when K. mikimotoi was under OA stress,

which was due to the fact that increased activity of GPx and GR promoted the GSH cycle (Zhang et al., 2022). On the basis, we speculate

that despite OA may cause oxidative damage, the presence of CCMs

seem to benefit the growth of K. mikimotoi under higher CO2 levels

exposure, which may increase the competitiveness of K. mikimotoi and

promote its outbreak in a future ocean.

Allelopathy of macroalgae has been considered to be one of the most

potential mechanism of inhibiting algal growth, while the increasing OA

driven by global warming may interfere with its allelopathic effects

(Zhang et al., 2015).Previous studies showed that elevated CO2 alleviated the allelopathic effects of Ulva species on S. costatum and lowered

competitive advantage of Ulva over microalgae (Gao et al., 2019). Our

findings indicated that elevated CO2 level alleviated the allelopathic

effects of U. pertusa on K. mikimotoi, including decreased ROS production

and oxidative stress, which caused a significantly decrease in caspase-3

and -9 protease activity compared to co-culture system under original

condition. Moreover, the release of some secondary metabolites is the

key mean to inhibit microalgae through allelopathic effects in macroalgae, and phenolic compounds, terpenoids and fatty acids are regarded

as the most common types of allelochemicals, which may be influenced

with an increasing CO2 level (Li et al., 2021a). Lobophora rosacea，an

brown macroalgae, has been reported that OA changed its metabolic

pathways, and reduced the synthesis of some metabolites, such as

lobophorenols and several C21 analogues with allelopathic effects,

which reduced allelopathic effects on other marine organisms (Gaubert

et al., 2020). In this study, free fatty acids classed and contents in

U. pertusa were changed as the elevated CO2 level, and 13 fatty acids

with allelopathic effects were regulated as well. 4 and 9 of above fatty

acids were up- and down- regulated, which meant acidification treatment altered the synthesis of allelochemicals and ultimately reduced

allelopathic effects of U. pertusa on K. mikimotoi. We suggest that difficulties of inhibiting K. mikimotoi utilizing U. pertusa may increase as the

reduced allelopathic effects of U. pertusa, which is likewise one of the

potential factors contributing to the large-scale outbreaks of K. mikimotoi

in a future ocean. Our study comprehensively predicted the impact of

rising CO2 levels on the outbreak of K. mikimotoi and the possibility of

controlling K. mikimotoi using U. pertusa in the future, which provided

evidence of the interactive effects between HABs species and macroalgae under CO2 enrichment.

Generally speaking, our study established a methodological framework to comprehensively evaluate the impacts of rising CO2 levels on

HABs species and the allelopathy effects of macroalgae on HABs species,

which predicts the possibility to control K. mikimotoi using macroalgae

in the future and complements relevant researches on controlling HABs.

5. Conclusion

In this study, we examined the responses of growth, metabolites and

apoptosis in K. mikimotoi to U. pertusa exposure and different CO2 concentrations (420 and 1000 ppmv). High CO2 level promoted the growth

of K. mikimotoi though OA induced a degree of oxidative damage and cell

apoptosis. OA alleviated the allelopathic effects of U. pertusa on

K. mikimotoi, and was demonstrated by the changes of soluble protein

contents and enzyme activity in antioxidant system and apoptosis system. This study also indicated that high level of CO2 disturbed the

metabolism of free fatty acids with allelopathic effects in U. pertusa,

resulting in the decreased allelopathic effects on K. mikimotoi. In sum,

these data collectively supported that rising CO2 levels may exacerbate

outbreak risk of K. mikimotoi blooms in the future.

Supplementary data to this article can be found online at https://doi.

C. Wang et al.

Science of the Total Environment 926 (2024) 171688

10

org/10.1016/j.scitotenv.2024.171688.

CRediT authorship contribution statement

Chao Wang: Data curation, Methodology, Software, Writing –

original draft, Formal analysis, Validation, Visualization, Writing – review & editing. Renjun Wang: Conceptualization, Funding acquisition,

Supervision, Writing – review & editing. Lingna Meng: Data curation,

Formal analysis, Software, Validation, Visualization. Wenjing Chang:

Methodology, Software, Writing – review & editing. Junfeng Chen:

Writing – review & editing, Funding acquisition. Chunchen Liu:

Writing – review & editing. Yuhao Song: Writing – review & editing.

Ning Ding: Writing – review & editing. Peike Gao: Conceptualization,

Resources, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence

the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgements

This work was funded by National Natural Science Foundation of

China (31971503) and Undergraduate Teaching Reform Research

Project of Shandong Province (Z2022221). Moreover， thanks to Biotree Biomedical Technology Co, Ltd. (Shanghai, China) for their supporting of GC–MS assisted derivatization test platform and in-house

database of compounds.

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