Marine Pollution Bulletin 158 (2020) 111313

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Marine Pollution Bulletin

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

The first benthic harmful dinoflagellate bloom in China: Morphology and T

toxicology of Prorocentrum concavum
⁎
Jian Zoua,b,1, Qun Lia,b,1, Songhui Lua,b,c, , Yuelei Donga,b, Heng Chena,b, Chengzhi Zhenga,b,
⁎
Lei Cuia,b,
a
Research Center of Harmful Algae and Marine Biology, Jinan University, Guangzhou 510632, China
b
Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China
c
Southern Marine Science and Engineering Guangdong Laboratory, Zhuhai, China

A R T I C LE I N FO A B S T R A C T

Keywords: More frequent events and geographic expansion of benthic harmful algal blooms have been reported in recent
Benthic harmful algae bloom years. An unexpected bloom of benthic P. concavum occurred in Xincun Bay, Hainan Island, the South China Sea
Prorocentrum concavum was monitored in August 2018. Species identification, toxin analysis and toxicity test were conducted in the
Morphology study. Quantitative study revealed that P. concavum had a high cell density on the surface of substrates and in
Phylogenetic analysis
water column. The bloom forming species was identified based on the morphology and phylogeny. Toxin
Toxicity assessments
analysis indicated that there were no detectable DSP toxins either in algae or in shellfish samples. The result of
toxicity test revealed that the extracts of P. concavum caused the mortality of brine shrimp larvae (Artemia
salina). The results from this study may provide more insight into the rising threats of harmful dinoflagellate
blooms to marine benthic ecosystems.

1. Introduction
Benthic harmful algal blooms (BHABs) are emergent marine disaster
to affect benthic ecosystem (Berdalet et al., 2017; GEOHAB, 2012).
Differed with the planktonic blooms, BHABs are induced mainly by
epibenthic dinoflagellates (Accoroni and Totti, 2016; Litaker et al.,
2010) and negatively altering marine benthic ecosystem (Accoroni
et al., 2016; Mangialajo et al., 2017). The most reported harmful
benthic dinoflagellates are Gambierdiscus, which are producers of toxin
ciguatera (Chan, 2015; Litaker et al., 2010; Russell and Egen, 1991),
and Ostreopsis, which are origin of ovatoxins and its derivatives (Amzil
et al., 2012; Tibiriçá et al., 2019).
The genus Prorocentrum, one of the largest groups in dinoflagellates,
was established in 1834 by Ehrenberg with Prorocentrum micans as type
species (Ehrenberg, 1834). So far 75 species has been identified, of
which around 30 species are associated with benthic habitats
(Hoppenrath et al., 2013; Lim et al., 2019; Rodríguez et al., 2018).
Some presumable epibenthic species (e.g., P. arabianum Morton & Faust
(=P. concavum Fukuyo)) are tychoplanktonic, which can also be col-
lected from the plankton (Morton et al., 2002). So far ten species from
Prorocentrum has been reported to produce diarrhetic toxins, okadaic
acid (OA) and the methyl derivatives dinophysistoxins (DTXs), of which
nine are benthic (An et al., 2010; Holmes et al., 2001; Hoppenrath
et al., 2013; Luo et al., 2017; Murakami et al., 1982; Nascimento et al.,
2016). There were also reports from some species of Prorocentrum for
production of various bioactive compounds (Faust and Gulledge, 2002).
Though Prorocentrum is one group of species for production of toxins
responsible for diarrhetic shellfish poisoning (DSP) in humans through
food train, reports of toxic blooms and DSP incidents related to benthic
Prorocentrum species were scarce (Foden et al., 2005; Gayoso et al.,
2002). It is partly because of the difficulty to detect the outbreaks of
toxic Prorocentrum species in the benthic nature.
P. concavum is one of the toxic Prorocentrum species (Dickey et al.,
1990; Hu et al., 1992). It was originally described by Fukuyo (1981) in
French Polynesia, New Caledonia and Ryukyu Islands and reexamined
by Mohammad-Noor et al. (2007). This species was synonymized as P.
arabianum (Mohammad-Noor et al., 2007) and P. faustiae (Chomérat
et al., 2019). The production of OA by P. concavum was first reported in
a culture strain from Caribbean (Dickey et al., 1990). The production of
OA and diol ester derivatives of OA has also been confirmed from ex-
tracts of culture strains of P. concavum (Hu et al., 1992). Some studies
also reported that P. concavum had cytotoxic, ichthyotoxic, and hae-
molytic activity, which were lethal to mice (Morton et al., 2002;
Yasumoto et al., 1987). However, it seems that the toxin productions

⁎
Corresponding authors at: Research Center for Harmful Algae and Marine Biology, Jinan University, Guangzhou 510632, China.
E-mail addresses: lusonghui1963@163.com (S. Lu), leicui@jnu.edu.cn (L. Cui).
1
These authors contributed equally.

https://doi.org/10.1016/j.marpolbul.2020.111313
Received 7 December 2019; Received in revised form 19 May 2020; Accepted 21 May 2020
Available online 17 June 2020
0025-326X/ © 2020 Elsevier Ltd. All rights reserved.
J. Zou, et al.
and the toxicity effects of P. concavum are probably strains specific. A
recent study on P. concavum revealed that there were no OA, DTX-1 and
DTX-2 detected in a tropical strain from Australia (Verma et al., 2019).
The toxin analysis of four strains of P. concavum collected from tropical
waters of Hainan Island, China indicated that all strains did not produce
detectable level of OA and DTX-1 (Luo et al., 2017).
There were numerous studies which revealed high diversity of
Prorocentrum in tropical benthic reef ecosystems (Irola-Sansores et al.,
2018; Lim et al., 2019; Verma et al., 2019). Previous study revealed that
there were seven Prorocentrum species, P. lima, P. rhathymum, P. con-
cavum, P. cf. emarginatum, P. fukuyoi, P. cf. maculosum (synonymized
with P. caipirignum by Nascimento et al. (2017)) and P. panamense have
been described from the northern South China Sea, of which OA was
detected in all strains of P. lima and P. caipirignum (Luo et al., 2017).
During our routine benthic dinoflagellate survey conducted on
tropical Hainan Island, China in 2018, a high density of P. concavum
bloom on surface of the substrates was encountered in Xincun Bay,
southeast of Hainan Island. The bloom was the first identified benthic
bloom in China. In this paper, the benthic bloom was described. The
detailed morphological observations of the bloom forming species, to-
gether with phylogenetics have been studied. The environmental
parameters where the bloom occurred were monitored. The culture
strain of the species was succeeded established and the toxicity of the
species was tested.
2. Materials and methods
2.1. Site description and sample collection
Xincun Bay is a tropical lagoon located in the southeast of Hainan
Island, South China Sea (Fig. 1). The bay (area of 13.1pkm2) is almost
closed, with only one narrow canal connected to the open sea (Yang and
Yang, 2009). The substrata of Xincun Bay mainly contains seagrasses,
macroalgae, silt, and sand, which are suitable for the growth of benthic
dinoflagellates (Fig. 2). In this study, samples were collected during the
benthic dinoflagellate bloom at site 1, where there are lots of seagrasses
(Thalassia hemprichii and Enhalus acoroides) and seaweed (Ulva lactuca).
Site 2 located in the mangroves without the presence of the bloom, was
designated as the reference site. Triplicate water samples were collected
using 100 ml sample bottles near the benthic substrates. The macro-
phyte samples were collected in three replicates at a depth of 0.4 m. The
epibenthic microalgae were washed several times using fresh seawater
to ensure they were entirely separated from the macrophyte. Some live
cells were transferred into a polycarbonate bottle and others reserved in
the 15 ml centrifuge tubes. After the tubed samples were preserved in a
1.5% Lugol's solution, the fresh seaweed was weighed. In addition, we
collected the field benthic microalgae assemblages and two shellfish
(Callista erycina and Gafrarium pectinatum) in the study area.
2.2. Hydrographic and nutrient parameters
The water temperature, pH, and salinity were measured using a YSI
meter (YSI-6600; YSI Incorporated, USA). The concentrations (three
replicates) of total nitrogen (TN), total phosphate (TP), nitrate (NO3-N),
nitrite (NO2-N), ammonium (NH3−N), phosphate (PO4-P), and silicate
(Si) were measured using spectrophotometry according to the methods
of Sagi (1966), Murphy and Riley (1962), Bendschneider (1952), Ebina
et al. (1983), and Mullin and Riley (1955), respectively.
2.3. Cell counting and morphological observations
Epibenthic dinoflagellates were identified and counted using an
inverted microscope (ECLIPSE TE 2000-U, Nikon, Japan) at 100×
magnification. The identification of P. concavum was based on the
morphology of the periflagellar area and dimensions. Other benthic
dinoflagellates were differentiated into five genera (Prorocentrum,
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Marine Pollution Bulletin 158 (2020) 111313
Ostreopsis, Gambierdiscus, Coolia, and Amphidinium) based on their
morphologic characteristics. The final bloom density data were ex-
pressed as cells g−1 wet weight for the seaweed.
Live P. concavum cells were surveyed and photographed under a
fluorescence microscope (Olympus BX 61, Olympus Corporation,
Tokyo, Japan) coupled with a QImaging Retiga 400R digital camera
(QImaging, Surrey, BC, Canada). Over forty P. concavum cells were
measured at 400× magnification using Image-Pro Plus v. 6.0 (Media
Cybernetics, Rockville, MD, USA). The cells stained with SYBR Green
were used to observe the shape and location of the nucleus and chlor-
oplasts. To collect the cells for scanning electron microscopy (SEM), a
1 ml exponential culture was centrifuged in a Sorvall Biofuge Primo R
(Thermo Fisher Scientific, Waltham, MA, USA) at 5000 × g for 2 min at
25 °C. The supernatant was removed, and the pellet was cleaned with
the filtered seawater. The pellet was fixed, dehydrated, and then coated
as described by Zhang et al. (2015). Then, the samples were photo-
graphed with a scanning electron microscope (Zeiss ULTRA™ 55, Carl
Zeiss Inc., Oberkochen, Germany).
2.4. PCR amplifications and phylogenetic analyses
A unialgal strain of P. concavum (HN437) was isolated from the live
samples using the methods of Luo et al. (2017). Isolated P. concavum
cells were grown in filtered seawater with the L1 medium (Guillard and
Hargraves, 1993). Unialgal cultures were incubated at 25 °C under a
12/12 h light/dark regime with an irradiance of 100 μmol pho-
tons m−2 s−1. The exponential cells of P. concavum were centrifuged at
10,000 ×g for 2 min at 4 °C. The DNA of P. concavum was extracted and
amplified as described Luo et al. (2017). The D1-D3 regions of large
subunit (LSU) was amplified using general primers (D1R: 5′-ACCCGC
TGAATTTAAGCATA-3′, D3B: 5′-TCGGAGGGAACCAGCTACTA-3′)
(Scholin et al., 1994). The PCR procedure was as follows: an initial
denaturation step at 94 °C for 4 min; 36 cycles of denaturation at an-
nealing at 94 °C for 20 s, annealing at 56 °C for 30 s, and extension at
72 °C for 45 s; and a final extension of 7 min at 72 °C. In addition,
internal transcribed spacer (ITS) was amplified using the general pri-
mers (ITS1F: 5′-TCGTAACAAGGTTTCCGTAG-3′, ITS1R: ATATGCTTA
AGCTCAGCGGG) (Pin et al., 2001). PCR conditions composed 3 min at
94 °C followed by 40 cycles of 30 s each at 94 °C, 30 s at 56 °C, 1.5 min
at 72 °C, and a final extension of 10 min at 72 °C. Sequencing was
conducted in both directions, using an ABI PRISM 3730XL (Applied
Biosystems, Foster City, CA, USA). The partial LSU and ITS sequences of
P. concavum from Xincun Bay were deposited into the GenBank data-
base under the accession numbers MN698951 and MN699564, re-
spectively. The LSU and ITS sequences were aligned using the multiple
sequence alignment program (https://www.ebi.ac.uk/Tools/msa/
muscle/). The aligned results were inputted into maximum likelihood
(ML) analyses using RAxML-HPC2 and XSEDE v. 8.2.10 on the CIPRES
website (https://www.phylo.org/). In addition, the best models of the
LSU and ITS sequences selected by PAUP (version 4.0) were SYM + G
and Bayesian Inference (BI), respectively, for which MrBayes (version
3.0) was used and run for 1,000,000 generations with sampling every
100 generations.
2.5. Extraction and detection of DSP toxins
Field samples of benthic microalgal assemblages attached to mac-
rophytes were concentrated and collected from Xincun Bay. The con-
centrated samples were centrifuged at 10,000 ×g for 10 min. Then,
10 ml of methanol was added to the pellets, before being ultrasonicated
thrice, for 20 min each time. The cells slurry was observed in an in-
verted microscope and centrifuged at 10,000 ×g for 15 min. Samples of
shellfish were collected from sampling site 1. Wet tissue samples of
Callista erycina and Gafrarium pectinatum (100 g each) were individually
homogenized using a T25 Ultra Turrax mixer at 24,000 rpm (IKA
Works, Wilmington, NC, USA). The homogenates were extracted and
J. Zou, et al.
Fig. 1. Sampling sites in Xincun Bay, South China Sea. Site 1 is in the seagrass bed; and Site 2 is in the mangroves, designated as the reference site.
purified according to the methods of Liu et al. (2019). To remove the
disturbance of DSP toxin analogues, 1 ml of each pure extracted solu-
tion was hydrolysed by 125 μl of 2.5 mol l−1 NaOH at 76 °C for 40 min.
Then, the solutions were neutralized by adding 125 μl of 2.5 mol ml−1
HCl.
For the laboratory experiment, P. micans (strain: HSJ19) and P. lima
(strain: XS326), obtained from Algal Culture Collection, Research
Center of Harmful Algae and Marine Biology, Jinan University,
Guangzhou, China, were selected as the negative and positive control,
respectively. Three litre mid-exponential cultures of P. concavum, P.
micans, and P. lima were individually cultivated and extracted ac-
cording to the methods of Luo et al. (2017). The field and cultured
pellets of P. concavum, P. lima, and P. micans were discarded and the
supernatants were collected and dried with N2. Dried algae sediments
were suspended separately in 5 ml of methanol and filtered with a spin-
filter (0.22 μm pore-size; Millipore Ultrafree, Eschborn, Germany).
Three lipophilic DSP toxins (OA, DTX-1, and DTX-2) of P. concavum, P.
micans, P. lima, and two shellfish were detected using liquid
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Marine Pollution Bulletin 158 (2020) 111313
chromatography-tandem mass spectrometry (LC-MS/MS), as described
by Liu et al. (2019). The detection limits of OA, DTX-1, and DTX-2 were
1.53, 3.04, and 1.34 ng ml−1, respectively.
2.6. Toxicity assessments
Dried brine shrimp (Artemia salina) cysts were incubated in filtered
artificial seawater (pore size: 0.22 μm; salinity: 30 ± 1 ppt) for 24 h.
Then, the cysts were aerated and maintained at 25 ± 1 °C,
100 μmol photons m−2 s−1, under a 12 h light: 12 h dark cycle. The
brine shrimp larvae were transferred into 12-well tissue culture plates
for the toxic bioassay. The toxicity of the field and cultured P. concavum
samples were examined using bioassays with brine shrimp larvae. The
concentration series of the treatment was 2.5 × 102, 2.5 × 103,
2.5 × 104, 5 × 104, and 2.5 × 105 cells ml−1. The 4 ml algal ex-
tractions were dried with N2 and suspended using 1% Tween-80 dis-
solved with phosphate-buffered solution for the toxicity test. The arti-
ficial seawater, solvent, and negative and positive controls consisted of
J. Zou, et al.
Fig. 2. Habitat during the P. concavum bloom in Xincun Bay, South China Sea. A, B, C, and D exhibit the seagrass bed, Enhalus acoroides, Thalassia hemprichii, and Ulva
lactuca, respectively.
filtered seawater, 1% Tween-80, and cultured P. micans and P. lima,
respectively. The concentrations of the solvent controls were diluted
according to the concentrations of the algal lysate solution. The con-
centrations of the negative and positive controls were similar to those of
the treatment. Twenty brine shrimp larvae were assigned to individual
wells of 12-well tissue culture plates and 2 ml of filtered seawater
(salinity: 30 ppt) was added for the toxicity test (48 h test). They were
maintained at 25 ± 1 °C, 100 μmol photons m−2 s−1, under a 12/12 h
light/dark cycle. The mortality and abnormality rates were recorded at
48 h. Deceased individuals were identified by a lack of movement in the
larvae appendages. Abnormal individuals were classified by abnormal
swimming behaviours or immobility (Leung et al., 2017).
2.7. Statistical analysis
The concentrations that led to a mortality of 50% of the field and
cultured P. concavum were counted using a sigmoidal-log mode curve
formed by Origin (version 9.60) (OriginLab software, USA). The sta-
tistical results were analysed using IBM SPSS Statistics 25 (IBM cor-
poration, Armonk, NY, USA).
3. Results
3.1. Bloom detection
The benthic Prorocentrum bloom was observed by chance on 27th
August 2018 in the south of Xincun Bay, Hainan Island. During the
bloom period, the seagrasses and macroalgae were covered with nu-
merous Prorocentrum cells (Fig. S1). Among them, the density of P.
concavum on T. hemprichii was the highest (3.0 × 105 cells g−1 wet
weight), followed by E. acoroides (2.2 × 105 cells g−1 wet weight),
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Marine Pollution Bulletin 158 (2020) 111313
while U. lactuca was the lowest (3.2 × 104 cells g−1 wet weight)
(Fig. 3). Similarly, the water column of the seaweed beds contained a
high amount of P. concavum cells (1.7 × 104 cells l−1). The total ratios
of P. concavum were 93.5%, 99.7%, 97.0%, and 99.7% in the T. hem-
prichii, E. acoroides, Ulva lactuca and water column samples, respec-
tively. In addition to P. concavum, four potentially toxic benthic dino-
flagellates (P. rhathymum, Coolia spp., Ostreopsis spp., and Amphidinium
spp.) were found on these substrata and in the water column. As shown
in Table 1, the water temperature, pH value, and salinity were 28.0 °C,
8.40, and 31.92 psu in the bloom area, respectively. Moreover, all of the
nutrient concentrations were lower in the bloom area than those in the
non-bloom area.
3.2. Morphology of P. concavum
Exhibiting the typical morphological characteristics of P. concavum,
the organism was symmetrically oval and had two pusules (Pu) situated
towards the anterior of the cells (Fig. 4A and B). The length (L) and
width (W) of the cells were 43.5–55.6 (mean = 48.84 ± 2.7 μm,
n = 41) and 34.3–45.8 μm (mean = 40.95 ± 2.5, n = 41), respec-
tively. In addition, the L/W ratio ranged from 1.11 to 1.30. The
chloroplasts (Chl) were well-distributed with central pyrenoids (P), as
shown in Fig. 4C. The oblong nucleus (N) was located in the posterior of
the cell (Fig. 4D). SEM micrographs of P. concavum displayed the
foveate thecal plates of the cells (Fig. 4E, F, and G). The plates were
ornamented with numerous depressions that decorated many pores
(Fig. 4H). The anterior V–shaped periflagellar area consisted of a fla-
gella pore and accessory pore, as well as nine platelets (1a, 1b, 2, 3, 4,
5, 6, 7, and 8), according to the periflagellar descriptions of Hoppenrath
et al. (2013) (Fig. 4I).
J. Zou, et al. Marine Pollution Bulletin 158 (2020) 111313

Fig. 3. Abundance of benthic dinoflagellates on the seagrasses, on the macroalgae, and in the water column during the bloom period. The unit of abundance for P.
concavum, Thalassia hemprichii, and Enhalus acoroides is cells g−1 wet weight and the unit of abundance in the water column is cells l−1.

Table 1 3.4. Detection of toxins causing DSP

Features of the bloom in Xincun Bay.
Parameters Site 1 (bloom area) Site 2 (non-bloom area)
LC-MS/MS experiments were conducted on the extracts of the field
and cultured samples using external calibration. There were no obvious
Temperature (°C) 28.0 – peaks observed in the extracts of the field and cultured P. concavum
Salinity (psu) 31.92 – samples (Fig. S3). Similarly, no DSP toxin signals were detected in the
pH 8.40 –
NO3-N (μmol l−1) 1.04 ± 0.04 1.90 ± 0.14
shellfish samples (C. erycina and G. pectinatum) or cultured P. micans.
NO2-N (μmol l−1) 0.02 ± 0.00 0.13 ± 0.04 However, OA and DTX-1 were detected in P. lima. The concentrations of
NH3-N (μmol l−1) 12.69 ± 1.83 15.77 ± 0.97 OA and DTX-1 for P. lima were 1871.31 and 80.88 pg cell−1, respec-
PO4-P (μmol l−1) 0.90 ± 0.11 1.19 ± 0.02 tively.
SiO3-Si (μmol l−1) 9.03 ± 1.22 12.04 ± 0.46
TN (μmol l−1) 17.74 ± 3.71 22.44 ± 0.40
TP (μmol l−1) 0.718 ± 0.22 0.83 ± 0.17 3.5. Toxicity of P. concavum lysate to brine shrimp

3.3. Molecular phylogeny
To determine the phylogenetic position of P. concavum HN437, 69
and 52 sequences of LSU and ITS of the genus Prorocentrum were ob-
tained from the GenBank, respectively. The LSU sequence of Adenoides
eludens ADE15 France (LC002847) and ITS sequence of Karena brevis
CCMP719 USA (AF352827) were used for outgroups. Phylogenetic trees
inferred from the ML and BI methods were constructed for the nu-
cleotide sequences of LSU and ITS, respectively (Figs. 5 and S2). The
phylogenetic tree based on LSU sequences revealed two main clades (A
and B) (Fig. 5). The clade A consisted of 13 species, which consisted of 6
kinds of benthic species (P. elegans, P. fukuyoi, P. scupltile, P. emargi-
natum, P. tsawwassenense and P. rhathymum) and 7 kinds of planktonic
species (P. dentatum, P. minimum, P. triesinum, P. koreanum, P. micans, P.
texanum and P. gracile). Eleven species of Prorocentrum were consisted
of the clade B, which were benthic species except for P. playfairi. P.
concavum, which branches into three independent sub-branches, shares
a close ancestry with P. leve. The first sub-branch consisted of the strains
IFR12–251 (MG701855) and NMN013 (EF566744), isolated from
Martinique and Malaysia, respectively. The HN437 P. concavum strain
and the four P. concavum strains isolated from Hainan Island
(KY010227, KY010228, KY010226, and KY010229) comprised the
second sub-branch. The last sub-branch included other strains isolated
from China (AJ567464), Malaysia (EF566751), and the Arabian Sea
(EF566752). Moreover, the phylogenetic analysis results based on ITS
regions were consistent with the phylogenetic tree based on LSU se-
quences. The P. concavum strain HN437 was the sister clade of the other
four P. concavum strains (KY010227, KY010228, KY010226, and
KY010229) isolated from Hainan Island (Fig. S2).
A lethal effect was detected in the field and cultured samples (P.
concavum, P. lima, and P. micans), following the concentrations of
2.5 × 102, 2.5 × 103, 2.5 × 104, 5 × 104, and 2.5 × 105 cells ml−1
(Fig. 6). Brine shrimp larvae mortality rates of over 50% were observed
for the field samples and cultured P. concavum in the concentrations of
2.5 × 104, 5 × 104, and 2.5 × 105 cells ml−1. For cultured P. lima and
P. micans, high mortality rates (> 80%) were observed for the treat-
ment with P. lima, only at the of concentration of 2.5 × 105 cells ml−1.
Based on the 48 h treatment of the field samples and cultured P. con-
cavum and P. lima, mortality rates of 50% (LC50 value) were estimated
using sigmoidal log-response curves (Fig. 7). The 48 h-LC50 values were
1.50 × 104 cells ml−1 for the field samples, 1.54 × 104 cells ml−1 for
the cultured P. concavum, and 5.34 × 104 cells ml−1 for the cultured P.
lima.
4. Discussion
4.1. The first P. concavum bloom in Chinese coastal waters
In this study, the greatest density of P. concavum was
3.0 × 105 cells g−1 wet weight on T. hemprichii. Simultaneously, P.
concavum on E. acoroides and U. lactuca also had a high density
(2.2 × 105 cells g−1 wet weight and 3.2 × 104 cells g−1 wet weight,
respectively). The density of P. concavum in Xincun Bay was much
higher than other Prorocentrum blooms of previous studies (e.g. the P.
lima blooms (approximately 4.5 × 104 cells g−1 wet weight) on mac-
roalgae in the French coast and the P. lima (1.0 × 104 and
3.0 × 104 cells g−1 wet weight, respectively) on Phaeophyta in
Caribbean Sea and on Thalassia in Gulf of Mexico) (Blanfuné et al.,
2015; Delgado et al., 2006). The average ratio of P. concavum

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J. Zou, et al. Marine Pollution Bulletin 158 (2020) 111313

Fig. 4. Light and scanning electron micrographs of P. concavum. (A and B) Light microscopy (LM); (A) a live field cell showing the shape of the cell and pusule, and
(B) a live cultured cell showing the shape of the cell. (C and D) Fluorescence LM; (C) a cell showing the location of the pyrenoids (P) and arrangement of the
chloroplasts (Chl), and (D) a cell stained with SYBR Green exhibiting the shape and location of the nucleus (N). (E–I) SEM; (E and F) the right and left valve showing
the V-shaped periflagellar area, countless depressions, and thecal pores, (G) the intercalary band showing the transverse striations, (H) the details of the thecal pores,
and (I) the details of the periflagellar area showing nine platelets, the flagella pore (fp), and the accessory pore (ap). Scale bars: 10 μm (A–G), 1 μm (H and I). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

abundance was 96.7% in the benthos and even reached 99.7% in the
water column. Additionally, we found that all of the nutrient con-
centrations were lower in the bloom area than in the non-bloom area,
indicating that the numerous P. concavum exhausted lots of nutrients
during the bloom. Thus, it is rare that the P. concavum bloom in Chinese
coastal waters had a high density and amount. Although the P. ara-
bianum (sym. P. concavum) bloom was reported previously in the water
column in the Gulf of Oman, 1995, this study did not concern the
concentration and hazard of the bloom (Morton et al., 2002). Besides, P.
arabianum was found in water column and was originally reported as a
planktonic species. As other benthic dinoflagellates (e.g. Ostreopsis), the
cell may detach from the substrate to water column by the hydro-
graphic conditions such as tide, wave and turbulence (Accoroni and
Totti, 2016). Thus, the same phenomenon could exist in P. concavum
and Prorocentrum other species. Due to occasional blooms and diffi-
culties in sampling, there were little reports paying attention to benthic
Prorocentrum so far. As one of the main results in present study, the
benthic P. concavum bloom was firstly reported in the substrate and
water column in Xincun Bay. The P. concavum bloom has not been
noted as present in China before and the abundance of the causative
species had not previously been reported anywhere in the world during
blooms as well. Therefore, it need further attention that if this benthic
P. concavum bloom occurred again in this region.

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J. Zou, et al. Marine Pollution Bulletin 158 (2020) 111313

Fig. 5. Phylogenetic tree for the genus Prorocentrum based on the LSU nucleotide sequences, using Bayesian inference (BI) and maximum likelihood (ML) methods.
The outgroup is Adenoides eludens ADE15 France (LC002847). The numbers above the branches refer to the ML bootstrap values (left) and Bayesian posterior
probability values (right). The black dots represent support values equal to 100/1.00. The values are > 50 and 0.8 for the ML bootstrap analysis and Bayesian
posterior probabilities, respectively. The scale bar represents the substitutions per site.

7
J. Zou, et al.
Fig. 6. Toxicology of the field and cultured samples (P. concavum, P. lima, and P. micans) to brine shrimp (48 h treatment). Solvent (SC) and filtered seawater (SW)
were provided for eliminating the effects of 1% Tween80 and artificial seawater.
Fig. 7. Sigmoidal-log mode curve stating the toxicity of the field and cultured
samples (P. concavum and P. lima) to brine shrimp (48 h treatment).
4.2. Identification of P. concavum
P. concavum was identified based on the morphology and phylogeny
in this study. Both the field specimens and cultured P. concavum were
generally consistent with the original morphological descriptions that
this species possessed a wide V-shaped periflagellar area (1a, 1b, 2, 3, 4,
5, 6, 7 and 8) and many depressions distributed in the thecal plates
except the center (Fukuyo, 1981). In the descriptions by Mohammad-
Noor et al. (2007), P. concavum had been reported as possessing two
types of pores, however, only one type of pore was distributed into the
depressions and these pores had a different diameter in the present
study. There were no pores in the margins of P. concavum and the
periflagellar area was divided into nine platelets, as platelet 1 was se-
parated into platelets 1a and 1b. These results were found in the pre-
vious researches (Luo et al., 2017; Mohammad-Noor et al., 2007; Verma
et al., 2019). In addition, the morphology of field specimens had similar
characteristics with other Prorocentrum species (P. lima and P. hoff-
mannianum) that two obvious pusules were situated below the acces-
sory pore (Fig. 4A) (Zhou and Fritz, 1993). Visible pusule had not been
observed in the cultured cells and this phenomenon could be associated
with a change in the growth environment during the laboratory culture
8
Marine Pollution Bulletin 158 (2020) 111313
(Calado and Hansen, 1999; Hansen et al., 2000). According to the
phylogenetic trees, the species P. concavum branched with the species P.
faustiae, P. foraminosum and P. leve (Figs. 5 and S2), which was con-
sistent with the previous phylogenetic results (Luo et al., 2017; Verma
et al., 2019). P. concavum strain HN437 closely formed a sister clade
with another P. concavum strains (DS4C10 and DS4E11) from Lingshui,
Hainan Island which was closed to this bloom area. The P. concavum
strains of Hainan Island were separated with other strains from Marti-
nique, Malaysia, and the Arabian Sea. This result supports the view that
Prorocentrum species from different geographical positions may have
morphological differences (Herrera-Sepúlveda et al., 2015; Hoppenrath
et al., 2013; Zhang et al., 2015). In the original descriptions by Morton
(1998), strain NMN013 was a new species P. faustiae which was first
found in Heron Island, Australia. However, some studies suggested that
P. faustiae was synonymized as P. concavum due to that the two species
were extremely similar in cell size, shape, pores and periflagellar area
(1a, 1b, 2, 3, 4, 5, 6, 7 and 8) (Chomérat et al., 2019; Hoppenrath et al.,
2013). Our phylogenetic analysis also revealed that the strain NMN013
branched into an independent sub-branch with the eight P. concavum
strains, shared a close ancestry with the strain of P. concavum
(IFR12–251) (Fig. 5). Hence the two species P. concavum and P. faustiae
might be conspecific based on a combination of morphology and phy-
logeny.
4.3. Toxicity of P. concavum
The three DSP toxins (OA, DTX-1, and DTX-2) were not detected in
both the cultured and field P. concavum in this study, which indicated
that the strain HN437 of P. concavum isolated from Xincun Bay might
not contain the three toxins or could produce rare toxins. But it is in-
conclusive whether P. concavum could produce OA, DTX-1 and DTX-2
and there are controversial data in some researches so far. Several
previous study found the extract of P. concavum could produce OA and
its diol ester derivatives (Hu et al., 1992). Similarly, OA and DTX-1 was
found in P. faustiae (sym. P. concavum) by using the HPLC-fluorescent
method (Morton, 1998). However, P. concavum in the tropical Australia
was not detected OA, DTX-1 and DTX-2 were in recent research (Verma
et al., 2019). Luo et al. (2017) found that P. concavum strains in waters
near Xincun Bay did not produce the three DSP toxins, except for two of
which were detected extremely low concentration of OA
(< 0.031 fg cells−1) below detectable level. The strain-specific varia-
tion of toxin production may exist in P. concavum. Some report showed
that the toxin concentration of Prorocentrum could be influenced by the
cultured conditions and this phenomenon should be considered in the
J. Zou, et al.
toxin investigation of P. concavum (Aquino-Cruz et al., 2018). Besides
the reason for strains and growth conditions, possible misidentification
of strains may lead to the difference in toxin detection. For instance,
Dickey et al. (1990) and Lillian et al. (1997) reported that P. concavum
could produce OA, but the species was similar to toxic species P. hoff-
mannianum based on the later morphology and phylogeny. Though no
DSP toxins were detected in present study, the results of the toxicity to
brine shrimp larvae showed that algae lysates were toxic and lethal to
marine invertebrate larvae. The LC50 value of P. concavum was
1.5 × 104 cell ml−1, which was lower than some toxic dinoflagellate
species, such as Coolia malayensis and P. lima for brine shrimp larvae
was approximately 3 × 105 cell ml−1 (Leung et al., 2017) and
5.34 × 104 cells ml−1 (present study), respectively. Thus, the bloom
strain of P. concavum isolated from Xincun Bay may be velogenic. Si-
milarly, the previous research suggested that P. concavum from Oki-
nawa, Japan didn't produce detectable OA, but the species had pow-
erful ichthyotoxicity (Yasumoto et al., 1987). Morton et al. (2002) also
showed that P. concavum from the Gulf Oman, Arabian Sea could induce
death in mice because of cytotoxic and haemolytic activity, although
the type of toxin was not clear. Based on above mentioned, P. concavum
from Xincun Bay may contain uncertain toxin rather than DSP toxins,
which were lethal to brine shrimp larvae. Hence, it is worth noting that
this benthic P. concavum had potential harm for the benthic marine
ecosystem and public health. In addition, the type of toxin needed for
further analyses to demonstrate toxic mechanism of P. concavum in the
future.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.marpolbul.2020.111313.
Acknowledgements
This work was supported by Special Foundation for National
Science and Technology Basic Research Program of China
(2018FY100200 and 2018FY100100) and National Natural Science
Foundation of China (41876173 and 41576162).
Declaration of competing interest
The authors declare no competing financial interests.
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