Allelopathic inhibition of photosynthesis in the red tide-causing marine alga, Scrippsiella trochoidea (Pyrrophyta), by the dried macroalga, Gracilaria lemaneiformis (Rhodophyta)

    Allelopathic inhibition of photosynthesis in the red tide-causing marine alga, Scrippsiella trochoidea (Pyrrophyta), by the dried macroalga, Gracilaria lemaneiformis (Rhodophyta) Changpeng Ye, Heping Liao, Yufeng Yang PII: DOI: Reference:

S1385-1101(14)00050-1 doi: 10.1016/j.seares.2014.02.015 SEARES 1223

To appear in:

Journal of Sea Research

Received date: Revised date: Accepted date:

18 June 2012 19 February 2014 28 February 2014

Please cite this article as: Ye, Changpeng, Liao, Heping, Yang, Yufeng, Allelopathic inhibition of photosynthesis in the red tide-causing marine alga, Scrippsiella trochoidea (Pyrrophyta), by the dried macroalga, Gracilaria lemaneiformis (Rhodophyta), Journal of Sea Research (2014), doi: 10.1016/j.seares.2014.02.015

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ACCEPTED MANUSCRIPT Allelopathic inhibition of photosynthesis in the red tide-causing marine alga, Scrippsiella trochoidea (Pyrrophyta), by the dried macroalga, Gracilaria lemaneiformis (Rhodophyta)

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Changpeng Ye *, Heping Liao, Yufeng Yang *

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Institute of Hydrobiology, Jinan University, Key Laboratory of Eutrophication and

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Red Tide Control, Education Department of Guangdong Province, Guangzhou 510632, Guangdong Province, China

* Author for correspondence E-mail: [email protected]; [email protected] numbers: +86 020 85220239

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Abstract

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The red tide-causing microalga, Scrippsiella trochoidea was co-cultured with different

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quantities of dried macroalga Gracilaria lemaneiformis under laboratory conditions, to characterize the allelopathic inhibition effect of the seaweed on photosynthesis of

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the microalga. Photosynthetic oxygen evolution was measured, and chlorophyll a (Chl

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a) fluorescence transient O-J-I-P (O, J, I and P point in primary photochemisty reaction curve in photosystem II) curves associated with its specific parameters were

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determined. A concentration-dependent inhibition of S. trochoidea was observed when the dried seaweed was added. The rate of light-saturated maximum photosynthetic oxygen evolution (Pmax) was markedly decreased, and the O-J-I-P

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curve coupled with its specific parameters was reduced. The inhibitory effects of the macroalga on the microalga, according to the JIP-test (the relative fluorescence analysis based on O-J-I-P curve) and the activity of oxygen evolution, include a decrease in the number of active reaction centers, the blocking-up of the electron transport chain, and the damage to the oxygen-evolving complex. This study suggests that dried G. lemaneiformis is effective in inhibiting photosynthesis of S. trochoidea, and could thus be a potential candidate for mitigating S. trochoidea blooms. Keywords: Eutrophication; Harmful algal blooms; Gracilaria lemaneifomis; Scrippsiella trochoidea; Allelopathy; Photosynthesis

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1 Introduction Nitrogen and phosphorus loading from industrial, agricultural and municipal sources

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accelerates eutrophication in coastal zones worldwide (Anderson, 2008).

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Eutrophication may cause explosive growth of phytoplankton including harmful algal

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blooms (HABs), which have significant detrimental effects on fishery resources, marine ecosystems and human health around the world (Heisler et al., 2008; Anderson, 2009). Climate induced changes can also act synergistically with anthropogenic

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nutrient enrichment to increase harmful algal bloom frequency and geographical extent (Hallegraeff, 2003; Paerl and Paul, 2012). Cosmopolitan species, Scrippsiella

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trochoidea (Stein) Loeblich III, is distributed mainly in neritic habitats from the tropical to cold-temperate seas (Kim and Han, 2000; Hold et al., 2001; Gu et al., 2008). Formerly deemed as a non-toxic species, S. trochoidea is a thecate

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dinoflagellate bloom microalga reported from Japan, Korea and China (Wand and

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Tang, 2008; Wand and Wu, 2009), and was reported recently also as a toxic species responsible for killing larvae of bivalve species (Tang and Gobler, 2012). In bloom

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conditions in the field, S. trochoidea was observed to produce smooth and calcareous or noncalcified cysts (Gu et al., 2008). Wild and cultured fish and shellfish kills have been reported to be associated with blooms of S. trochoidea in Australia (Hallegraeff,

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1992) and China (Qin and Zou, 1997). Therefore, there is a need to develop management and mitigation strategies to respond to HABs. Various physical approaches, such as light-shading and solar ultraviolet radiation (Sugawara et al., 2003; Chen et al., 2009), and chemical strategies (Sun and Choi, 2004; Lee et al., 2008) have been developed. However, the large-application of these methods is limited by high cost and the potential for ecological secondary pollution (Anderson, 1997; Wang et al., 2009). In contrast, biological controls using macroalgae such as Ulva pertusa and Gracilaria speices are found to mitigate HABs effectively. These species are are indigenous to the marine environment, easy to collect at low cost and environmentally benign (Jin and Dong, 2003; Tang and Golber, 2011; Wang et al., 2012).

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ACCEPTED MANUSCRIPT G. lemaneiformis, an edible red alga broadly distributed and intensively cultivated in the coastal areas of China, is an economically important alga for agar extraction (Xu and Gao, 2008) and extraction of other natural products with important

additive in aquaculture (Qi et al., 2010).

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bioactivity (Zheng and Gao, 2009; Yeh et al., 2012), and is also used as a food It has been shown that G. lemaneiformis

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and other macroalgae have an inhibitory effect on the growth of some HAB species whether they are added as fresh thalli, culture filtrate, water-soluble extract or dry powder (Wang et al., 2007; Nan et al., 2008; Oh et al., 2010).

These macroalgae can

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therefore mitigate the negative effects of HABs by varying the make up of the phytoplankton community, changing the dominant species and decreasing microalgal

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abundance (Zhou et al., 2006). Compared to the direct application of fresh thalli of macroalgae, the use of extracts of macroalgal allelochemicals, dried powder or

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shredded segments of macroalgae would be safer, because the concentrations could be

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controlled and applied without seasonal restriction. Despite a number of studies on growth of HABs, the photosynthetic inhibitory

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mechanisms by which dried seaweed may affect HABs remains unconfirmed. In this study, the inhibition of S. trochoidea photosynthesis by dried G. lemaneiformis is characterized. As photosynthesis is the primitive driving force of physiological and

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biochemical processes in photoautotrophs, characterizing the photobiological profile provides useful information about the mechanisms by which dried G. lemaneiformi acts as a potential algicide souse against blooms of S. trochoidea which cause red tide. 2 Materials and methods 2.1 Culture of the seaweed Fresh thalli of Gracilaria (Bory) Dawson were collected in April 2011 from the Nanao Island Cultivation Zone (116.6°E, 23.3°N), Shantou, Guangdong, China. Thalli were transported in 500 mL sterile bottles filled with sterile seawater (SSW) to the laboratory where they were rinsed thoroughly with 100mL SSW and treated with a mixture of penicillin, chloramphenicol, polymixin and neomycin at non-inhibiting concentrations after Nakanishi et al., (1996) and Jeong et al., (2000). The medium

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ACCEPTED MANUSCRIPT used for algal cultivation was prepared according to the method of Jin and Dong (2003). The pH and salinity were adjusted to 8.0 and 30‰, respectively. Treated thalli were placed in sterile bottles containing SSW and were allowed to adapt to the

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laboratory environment for 5 d before use in experiments. Nutrients were enriched in

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the culture medium weekly by adding 100 µmol L-1 of NaNO3 and 7µmol L-1 of

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NaH2PO4. Nutrient enrichment was stopped 1 week before the experiments. The temperature was kept at 20 ± 0.1 ºC on a 12:12 light:dark cycle. Illumination was provided by cool-white fluorescent lamps at 70 µmol photons m-2 s-1. G.

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lemaneiformis fresh thalli were always maintained axenically under laboratory conditions and the effect of the environmental bacteria was considered negligible.

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2.2 Culture of Microalga

S. trochoidea was cultured in modified f/2 medium (Guillard, 1975) at 20°C, 70 µmol m-2 s-1 under a 12h:12h LD cycle. The initial pH and salinity of the culture medium

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were adjusted to 8.0 ± 0.02 and 30‰, respectively. The flask containing microalga

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was shaken manually twice daily, and grown to exponential phase for use in the

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experiments. Cells were inoculated into 500 mL Erlenmeyer flasks containing fresh f/2-enriched seawater until the total volume was 300 mL. The initial cell density was 1.0 × 104 cells mL−1. The microalgal culture (monoculture) was used as a control

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throughout the experiment.

2.3 Experiments with microalga and dried G. lemaneiformis Fresh G. lemaneiformis was rinsed to remove salt and air-dried in a super-purifier clean bench (4 Foot Purifier Clean Bench, Labconco, Kansas City, MO, USA) at room temperature. The dried thalli were cut into small pieces and amounts from 0 to 3.0 g dry wt L-1 were added to flasks containing the microalga. Moncultures with only S. trochoidea was served as the control. Three replicates were prepared for each experiment, and experiments were monitored for 3 days. 2.4 Measurement of photosynthetic oxygen evolution and dark respiration rates On the first and third day, photosynthetic oxygen evolution rates of S. trochoidea were measured with a Clark-type oxygen electrode fitted with a DW3 chamber (Hansatech, Kings Lynn, Norfolk, England) kept at 20°C by a temperature controller (Cole-Parmer Instrument Co., Vernon Hills, IL, USA) connected to the water jacket 4

ACCEPTED MANUSCRIPT of the electrode. 50 mL samples of S. trochoidea with different amounts of dried G. lemaneiformis were centrifuged at 20°C and 2000 rpm in high speed refrigerated centrifuge (5804R, Eppendorf, Germany). The centrifuged sample was put in a

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reaction vessel filled with 2 mL seawater (pH 7.8) containing 6 mM NaHCO3 as the inorganic carbon source. Based on preliminary experiments, 6 mM NaHCO3 was

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determined to be the optimal concentration for obtaining maximal net photosynthetic O2 evolution (data not shown). Photosynthetic O2 evolution was measured for each sample continuously for 5 min at a light-saturated irradiance of 400 µmol photons m-2

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s-1 (halogen lamp, 220V, 150W, PHILIP, PAR, 400-700 nm). Photosynthetically active radiation (PAR) was measured with a Li-185B quantum sensor (Li-Cor, Lincoln, NE,

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USA). Dark respiratoration rates were determined by covering the chamber with a black cloth.

2.5 Measurement of chlorophyll fluorescence

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On the first and third days, Chl a fluorescence transients of S. trochoidea were

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measured at room temperature using a plant efficiency analyser (PEA, Hansatech

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Instruments, Norfolk, England) with an actinic light of 3000 µmol photon m-2 s-1 (Strasser et al., 1995). Illumination was provided by an array of six high-intensity light-emitting diodes (with a peak wavelength of 650 nm), which were focused on the

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sample surface to provide homogeneous illumination over an area of 4 mm in diameter. All samples were dark-adapted for 15 min before measurement. The fluorescence intensities at 50 µs, 300µs (K-step), 2 ms (J-step) and 30 ms (I-step) were denoted as F0, F300µs, FJ and FI, respectively, and Fm was assumed as the maximum fluorescence intensity (Strasser et al., 1995). The specific parameters were calculated according to the JIP-test (Appenroth et al, 2001). To carry out the JIP test, several extracted and technical fluorescence parameters calculated from the measurements of the polyphasic fluorescence transients are needed. They are: (1) the minimal fluorescence yield, F0 (the fluorescence intensities when all reaction centers are open), (2) the maximal fluorescence yield, Fm (the excitation intensity is high enough to ensure the closure of all reaction centers), (3) the initial slope at the beginning of the variable fluorescence transients theoretically at time zero), dV/dt0 5

ACCEPTED MANUSCRIPT [=4(F300µs-F0)/(Fm-F0)]; and (4) the relative variable fluorescence at phase J, VJ [=(FJ-F0)/(Fm-F0)]. According to the JIP test, the energy flux for absorption (ABS), energy flux for trapping (TR) and enegry flux for electron transport (ET) per

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photosystem II (PSII) reaction center (RC) are given by equations 1-3, respectively. The concentration of the PSII reaction centers (RC/CS, indicating the density of active

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reaction center, i.e. photosynthetic units) are is given by equation 4 or 9 and 10. Specific fluxes or specific activities of photosystem: ABS/RC = [(dV/dt0)/VJ]/[1-(F0/Fm)]

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TR0/RC = (dV/ dt0)/VJ

Density of reaction centrescenters:

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ET0/RC = [(dV/ dt0)/VJ]·(1-VJ)

RC/CS = [VJ/(dV/ dt0)]·[1-( F0/Fm)]·F0

(1) (2) (3)

(4)

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Quantum efficiencies or flux ratios:

φPo = TR0 /ABS = 1-( F0/Fm) = Fv/Fm

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(6)

ψ0 = ET0 / TR0 = (1- VJ)

(7)

ABS/CS0≈F0

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Phenomenological fluxes or phenomenological activities: (8) (9)

RC/CSm = φPo·(VJ/Fm)·(ABS/ CSm)

(10)

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RC/CS0 = φPo·(VJ/F0)·(ABS/ CS0)

Where ABS/ CS0 represents absorption flux per excited cross-section of sample (at t=0), indicating the quantity of antenna chlorophyll; RC/CS0, the amount of active PSII reaction centers per excited cross-section (at t = tF0); RC/CSm, the amount of active PSII reaction centers per excited cross-section (at t = tFM); ψ0, efficiency with which a trapped exciton can move an electron into the electron transport chain beyond the reduced primary bound plastoquinone (QA-) (at t=0); φPo (Fv/Fm), the maximum quantum yield or efficiency of primary photochemistry. 2.6 Determination of Chl a After measurement of photosynthetic oxygen evolution and dark respiration, the whole sample of S. trochoidea in the reaction vessel was collected in a 15ml tube and centrifuged for 10 min at 5000 rpm at 20 °C in the high speed freezing centrifuge 6

ACCEPTED MANUSCRIPT (5810R, Eppendorf, Germany).

The sediment was extracted in 4ml absolute

methanol for 24 h at 4°C in the dark. This extract was centrifuged at 5000 rpm for 10 min and analyzed for Chl a content with a scanning spectrophotometer (UV 530,

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Beckman Coulter, USA). The Chl a concentration was calculated according to Wellborn (1994).

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2.7 Data statistical analysis

Data were analyzed by two-way ANOVA followed by a multiple comparison using the least significance difference (LSD) test. Calculations and statistical analyses were

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performed with SPSS 13.0 for Windows. P-values of 0.05 were considered as a significant.

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3 Results

3.1 Inhibition by dried G. lemaneiformis of photosynthetic oxygen evolution and dark respiration of S. trochoidea.

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Dried fragments of G. lemaneiformi exhibited a strong algicidal effect on S.

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trochoidea. The maximum photosynthetic oxygen evolution rate (Pmax) was

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significantly inhibited (P<0.01), and a concentration-dependent inhibitory pattern was observed, both on the first and third day (Fig. 1).

Compared to the control

(monoculture with only microalga), the Pmax with 1, 2, or 3 g L-1 dried biomass of the

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macralga treatment was negative, indicating photoinhibition was coupled with photorespiration. The inhibitory effect on Pmax was concentration-dependant, with the Pmax values 2.8 times lower at 2 and 3 g L-1 dried biomass compared to the 1 g treatment (Fig. 1A). On the third day, the inhibition effect of 2 g L-1 dried biomass of seaweed against Pmax of S. trochoidea was significantly higher than that of 3 g L-1 dried sample (P<0.01) (Fig. 1B). Compared with the control, the dark respiration rate (Rd) of the microalga was significantly increased by addition of macroalga (P<0.01). The Rd with 1, 2, and 3 g L-1 dried G. lemaneiformis treatment increased by 2.5, 8.3 and 7.9 times, respectively, on the first day (Fig. 2A). On the third day, similarly to Pmax, 2 g L-1 dried biomass of seaweed showed a stronger inhibitory effect (P<0.01) against S. trochoidea than 3 g L-1 dried biomass (Fig. 2B). 7

ACCEPTED MANUSCRIPT 3.2 Inhibition by dried G. lemaneiformis on the photochemical processes of S. trochoidea. Figure 3 shows fluorescence induction curves for dark -adapted samples. The addition

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of dried G. lemaneiformis lowered the polyphasic chlorophyll fluorescence transients (OJIP) intensities of S. trochoidea both at day 1 and day 3, with a positvie correlation

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between the amount of dried biomass added and the amount of inhibition (Fig. 3). Compared with the Chl a fluorescence intensity of S. trochoidea on the first day, Chl a fluorescences for every treatment increased significantly by the third day, indicating

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that the allochemical inhibition of dried seaweed weakened with time. The same concentration-dependent inhibition pattern were found in tFv/Fm, RC/CS0,

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RC/CSm, ABS/CS0 andψ0 on the first day (Figs. 4A, 5A, 6A, 7A and 8A). On the third day, the difference between 1 g and 2 g L-1 dried biomass was not significant

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(P>0.05), while the difference between the higher dried biomass of 3 g L-1 and other

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treatments was still significant (P<0.01) (Figs. 4B, 5B, 6B, 7B and 8B). Thus, it

4 Discussion

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appears that the inhibitory effect of dried G. lemaneiformis receded over time.

Allelopathy is the biochemical interaction, both stimulatory and inhibitory, between primary producers or between primary producers and microorganisms (Molisch,

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1937). When S. trochoidea was cultured with different concentration of dried G. lemaneiformis, the fragments stayed on the bottom of flask and did not shade the microalga, leading to the assumption that allelochemicals inhibited the microalga in this study. In the present study, Pmax of S. trochoidea was strongly suppressed by dried G. lemaneiformis, and there was a clear concentration-dependent pattern, indicating the partial deactivation of the water-splitting system (or OEC – oxygen evolving complex) in S. trochoidea by the seaweed. By the third day, the inhibitory effect of 2 g L-1 dried biomass of seaweed against Pmax of S. trochoidea was significantly higher than that of 3 g L-1 dried sample. During cutting of the dried seaweed other substance might be released in addition to

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ACCEPTED MANUSCRIPT allelochemicals. These substances may have a chelating effect on allelochemicals, decreasing the allelopathic role of allelochemicals, with higher quantities of dried seaweed creating larger chelating effects. The inhibitory effect may therefore be a

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competion between release of allelochemical substances and chelating processes.

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Compared with 3 g L-1 dried biomass of seaweed, 2 g L-1 sample may relaese more

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allelopathiclly active substance than are chelated. Also, it has been found that during the decay process, a process which is similar to cutting the dried seaweed in this study, plants also release allelochemicals and other compounds to the environment, such as

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the nutrient phosphorous, which interfered with the allelopathic effects of allelochemicals (Hu and Hong, 2008; Poulson et al., 2010; Wang et al., 2012).

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The dark respiration rate (Rd) following the same pattern as Pmax, was boosted with the increased dried biomass of macroalga compared with control , indicating that

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more energy was required for the enzymatic antioxidant defense system in microalgae

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to protect against the allelopathic effect, though further studies should be conducted to analyze the mechanimsms by which such a defense system might operate.

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All oxygenic photosynthetic organism investigated so far show a polyphasic rise of of fluorescence transients during the first second of illumination, which are labeled as O-J-I-P (Strasser et al., 2000). O-J-I-P is a useful, non-invasive tool for the study of

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the photosynthetic apparatus, and more specifically the behavior of photosystem II (Srasser et al., 1999; 2000). This O-J-I-P polyphasic transient has been found to change its shape according to changes in environmental conditions (Srivastava and Strasser, 1997; Clark et al., 2000). Quantitative analysis of recorded O-J-I-P Chl a fluorescence transients, by means of the JIP-test, result in the calculation of several biophysical parameters, which contain information about the fluxes of photons, excitons, electrons and further metabolic events at a given physiological state, and which can be used to quantify PSII function to different stressors (refer to Strasser et al. 2000 for a review). In our experiment, the O-J-I-P Chl a fluorescence transients, coupled with several biophysical parameters in S. trochoidea was depressed by the addition of dried G.

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ACCEPTED MANUSCRIPT lemaneiformis. ABS/ CS0, absorption flux per excited cross-section of sample (at t=0) declined, indicating the quantity of antenna chlorophyll had decreased, so that not enough light was harvested to support the subsequent photochemical process. The

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concentration of the PSII reaction centers (RC/CS) decreased, indicating the density of active reaction center, i.e. photosynthetic units decreased, which reduced the

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photochemical transferring of harvested and excited photons. ψ0, efficiency with which a trapped exciton can move an electron into the electron transport chain beyond the reduced primary bound plastoquinone (QA-) (at t=0) was reduced, suggesting that

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photosynthetic electron transport was blocked, resulting in a decline in the energetic grouping between photosynthesis units. φPo (Fv/Fm), the maximum quantum yield or

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efficiency of primary photochemistry, also dropped, suggesting that photochemical flux of photons, excitons, electrons and further metabolic events in the photosystem of

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S. trochoidea were depressed.

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In this study, the main photosynthetic inhibition targets by G. lemaneiformis on S. trochoidea were a decrease in the quantity and size of antenna chlorophyll (reflected

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by the decrease of ABS/ CS0), the number of active reaction centers (decrease of RC/CS0 and RC/CSm); the photochemical efficiency in PSII (a decrease of Fv/Fm), the blocking of the electron transport chain (a decrease of ψ0), and the damage to the

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oxygen-evolving complex (a decrease of Pmax and increase of Rd). Tang and Gobler (2011) showed that dried Ulva lactuca was equally or more potent than fresh material, an observation correlated with the viewpoint that polyunsaturated fatty acids or organosulfur compounds are active allelopathic agents (Alamsjah et al., 2008). Dithiolane and trithiane compounds isolated and identified from Characean species were also found to have allelopathic effects on epiphytic diatoms and other phytoplankton (Wium-Andersen et al., 1982). The enhanced strength of the extracts also suggests that allelochemicals in cells were more concentrated than the portion released into the environment. Lu et al. (2011) isolated some secondary metabolites from G. lemaneiformis, such as glycolipid compounds, fatty acid compounds, amides, phenolic compounds and terpenoids. And by screening of allelochemicals, they found that the strongest allelopathy on the growth of the red tide alga, Skeletonema costatum 10

ACCEPTED MANUSCRIPT was linoleic acid (Lu et al., 2011). In our experiment, the dried G. lemaneiformis thalli was mechanically cut into small fragments, a process might enhance the production and/or release of allelochemicals (Reigosa et al., 1999).

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Our results show that dried G. lemaneiformis strongly suppressed the photosynthesis of S. trochoidea culture, and that there was a clear concentration-dependent

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relationship reflected in a decreased Pmax and O-J-I-P curve along with its specific parameters, such as the Fv/Fm, RC/CS0, RC/CSm, ABS/CS0 and ψ0, revealing that the oxygen evolution complex, reaction centre and electron transport were damaged

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and/or inhibited, which finally resulted in the decrease of photosynthesis in S. trochoidea. Zhu et al. (2010) also found that allelopathic polyphenols, pyrogallic acid

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and gallic acid, isolated from submerged macrophyte Myriophyllum spicatum were the main factors responsible for the inhibition of the microalga. The inhibitory

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mechanims was for the inhibition of PSII (from the oxygen-evolving

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complex to plastoquinone) and whole chain (from oxygen-evolving complex to the photooxidized chlorophyll ) activities of Microcystis aeruginosa, and a similar

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response pattern in photosynthetic parameters was observed in our experiments. The allochemical inhibitory effect of dried G. lemaneiformis on S. trochoidea was also time-dependent. As shown in the O-J-I-P curve and associated parameters for S.

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trochoidea, on the first day the difference between 1 g and 2 or 3 g dried biomass was significant, but on the third day the difference was insignificant. These results are consistent with those of Nan et al. (2008), who found that the culture filtrate of macroalga Ulva lactuca inhibited the growth of Skeletonema costatum in the first or second day, but growth of the diatom resumed in the following days, suggesting that the allelopathic compounds degrade over time. Long-term inhibition might therefore require continuous addition of allelogpathic compounds. As for the dried seaweed of Gracilaria lemaneiformis, the balance and/or competiton between the acitivity of algaecide and orgainic nutrient isolated from this organism, determine the growing or dying pattern of HABs. In our laboratory controlled experiment, the HABs was strongly suppressed to death on the third day after inoculation with the dried seaweed, indicating that the algaecide originated from 11

ACCEPTED MANUSCRIPT the macroalga, exhibited a significantly greater inhibited activity compared to its organised macromolecular nutrients promoting ability. Moreover, in order to strengthen the algae-inhibitory effect and weaken the potential adverse effects by the

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the dried seaweed, some specific semipermeable membrane bags can be used as container to load the dried seaweed fragments, through which only

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low-molecular-weight substances, such as linoleic acid can pass, while the organised macromolecular nutrients can not. Via this way, as used in other algicidal agent against the harmful algae (Han et al., 2011), the biomass of dried seaweed can be

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easly controlled during application of the algicide for prevention and mitigation of HABs. Though, further in-situ experiments should be performed to apparise the

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allelopathic effect of dried seaweed on HABs. 5 Conclusion

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The results of this study suggest that dried G. lemaneiformis had negative allelopathic

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effects on the photosynthesis of S. trochoidea and could thus be a potential algicide to control and mitigate S. trochoidea blooms. Still, for application point, more further

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experiment should be carried out in the future field work.

Acknowledgements:

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This work was supported by National Key Technology R&D Program (No. 2012BAC07B05), National Natural Science Foundation of China (No. 31370476; 31000240), Project on the Integration of Industry, Education and Research of Guangdong Province (No. 2012B091100341).

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Fig. 1. The maximum photosynthetic oxygen evolution rate (Pmax) of S. trochoidea

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(A) and third day (B). Data are the means±SD (n=3). Bars with the same letters are not significantly different (P<0.05).

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Fig. 2. The dark respiration rate (Rd) of S. trochoidea cultured with different dried biomass of G. lemaneiformis on the first day (A) and third day (B). Data are the

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Fig. 3. The O-J-I-P curves of S. trochoidea cultured with different quantities of dried

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biomass of G. lemaneiformis on the first day (A) and third day (B).

Fig. 4. The maximum photochemical efficiency of PSII (Fv/Fm) in S. trochoidea cultured with different dried biomass of G. lemaneiformis on the first day (A) and

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third day (B). Data are the means±SD (n=3). Bars with the same letters are not significantly different (P<0.05).

Fig. 5. The amount of active PSII reaction centers per excited cross section at t = 0 (RC/CS0) of S. trochoidea cultured with different amounts of dried biomass of G. lemaneiformis on the first day (A) and third day (B). Data are the means±SD (n=3). Bars with the same letters are not significantly different (P<0.05).

Fig. 6. The amount of active PSII reaction centers per excited cross section at t = tFM (RC/CSm) of S. trochoidea cultured with different amounts of dried biomass of G. lemaneiformis on the first day (A) and third day (B). Data are the means±SD (n=3). Bars with the same letters are not significantly different (P<0.05). 19

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Fig. 7. The absorption flux per excited cross sectionat t=0 (ABS/CS0) of S. trochoidea cultured with different amounts of dried biomass of G. lemaneiformis on the first day

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Fig. 8. The efficiency with which a trapped exciton can move an electron into the electron transport chain (ψ0) of S. trochoidea cultured with different amounts of dried

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Highlights: Dried Gracilaria lemaneiformis can inhibit photosynthesis of Scrippsiella trochoidea

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The inhibitory effect was both concentration and time-dependent

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