Who is the “murderer” of the bloom in coastal waters of Fujian, China, in 2019?*

CEN Jingyi , WANG Jianyan , HUANG Lifen DING Guangmao , QI Yuzao CAO Rongbo CUI Lei LÜ Songhui

1 College of Life Science and Technology, Jinan University, Guangzhou 510632, China

2 Key Laboratory of Aquatic Eutrophication and Control of Harmful Algae Blooms of Guangdong Higher Education Institutes, Guangzhou 510632, China

3 Department of Science Research, Beijing Museum of Natural History, Beijing 100050, China

4 Fujian Fishery Resources Monitoring Center, Fuzhou 350003, China

Received Jul. 8, 2019; accepted in principle Jul. 30, 2019; accepted for publication Sep. 8, 2019 © Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract On May 24-29, 2019, a bloom occurring in Pingtan coastal areas of Fujian Province caused mass mortality of cage-cultured fi sh ( Plectorhinchus cinctus and Pagrosomus major). During the bloom, two major causative organisms were present: Prorocentrum donghaiense (at a concentration of 1.46×10 7 cells/L) and an unknown naked dinofl agellate (4.58×10 6 cells/L). The naked dinofl agellate was isolated and cultured in this study, and its morphological features were examined using light microscopy and scanning electron microscopy. The large subunit (LSU) of the rRNA gene and the internal transcribed spacer (ITS) region of the naked dinofl agellate were also sequenced for fi eld bloom samples and lab culture strains (PT-A and PT-B). On the basis of its morphological characteristics and molecular sequences, the unknown naked dinofl agellate was identifi ed as Karlodinium digitat um. According to the phylogenetic analysis, the K arl. digitat um was most closely related to Karlodinium austral e and Karlodinium armiger, and the three species clustered into a single clade of Karlodinium with bootstrap/posterior probability values of 95%/0.99 and 86%/0.99 inferred from LSU and ITS sequences, respectively. Karl. digitatum was fi rst reported as Karenia digitata, a new harmful algal species bloomed in Hong Kong, China, in 1998. In present study, we gave a detailed morphological and phylogenetic description of Karl. digitatum and submitted the molecular sequences of this species to GenBank for the fi rst time.

Keyword: Karenia digitata; Karlodinium digitatum; harmful algal bloom; Ichthyotoxicity

1 INTRODUCTION

Harmful algal blooms are proliferations of algae that kill marine organisms, contaminate seafood with toxins, and/or cause ecological damage (GEOHAB, 2001). Karenia G. Hansen & Moestrup 2000, a harmful genus of unarmored dinofl agellates, was established following a molecular and morphological study of athecate dinofl agellates previously contained within the genus Gymnodinium (Daugbjerg et al., 2000; Gómez et al., 2005). Major characteristics of Karenia are the presence of a straight apical groove on the epicone and the possession of fucoxanthin or fucoxanthin-derived accessory pigments (de Salas et al., 2003; Bergholtz et al., 2006).

In the past few decades, Karenia blooms have occurred in many sea areas worldwide, such as the United States (Tester and Steidinger, 1997), New Zealand (Chang et al., 2001), Japan (Oda, 1935), Korea (Cho, 1981), China (Lu and Hodgkiss, 2004; Lu et al., 2014), Norway (Tangen, 1977), Chile (Clément et al., 2001), Australia (de Salas et al., 2004), and South Africa (Botes et al., 2003). Karenia species have become well known because the toxins they produced can cause animal mass mortalities, neurotoxic shellfi sh poisoning, and respiratory distress (Gentien, 1998; Heil and Steidinger, 2009; Brand et al., 2012).

Fig.1 Map of Fujian Province (left) showing sampling sites (right)

Blooms of Karenia species are also severe and frequent occurred in China. K. mikimotoi is widely distributed in Chinese coastal waters, and was the second-most devastating species in this country (Lu and Hodgkiss, 2004; Lu et al., 2014). According to the China Marine Disaster Bulletin, 2005-2017, the economic loss induced by K. mikimotoi blooms was approximately US$377 million in China (SOAC, 2005-2017). Notably, in 2012 alone, 12 consecutive blooms of K. mikimotoi occurred in the coastal waters of Zhejiang and Fujian provinces, and caused massive fi nancial losses exceeding US$330 million (SOAC, 2013; Li et al., 2017).

Currently, 12 described species have been identifi ed in the genus Karenia (Gómez, 2012), and only 4 species ( K. mikimotoi, K. brevis, K. digitata, and K. longicanalis) have been reported in Chinese coastal waters (Liu, 2008). Although only K. mikimotoi was highly concerned and clearly studied for its frequent bloom events in China, we have detected other species, eg. K. longicanalis, was co-occurred with K. mikimotoi in some blooms (Wang et al., 2018). Multi-species Karenia blooms were also observed in other foreign coastal waters (Haywood et al., 2004; Steidinger et al., 2008; Heil and Steidinger, 2009). Difference erent Karenia species have difference erent toxin profi les and contribute difference erently to fi sh or shellfi sh mortality (Brand et al., 2012). Therefore, it is necessary to perform a comprehensive and accurate identifi cation for the causative species, especially in a harmful and toxic algal bloom, and this is also useful for the bloom management and monitoring.

During May 24-29, 2019, a bloom occurred in Pingtan coastal areas of Fujian province. Two species were mainly occurred in the bloom, the fi rst dominated species was identifi ed as Prorocentrum donghaiense, with a concentration attached 1.46×107cells/L (Cen, unpublished), and the second-most dominant species, with a concentration as high as 4.58×106cells/L, was another unknown naked dinofl agellate. The bloom caused massive mortality of cage-cultured fi sh ( Plectorhinchus cinctus and Pagrosomus major) (reported by Fujian Provincial Department of Ocean and Fisheries, 2019). In this study, we will give a detailed identifi cation of the unknown dinofl agellate based on morphological observations and a molecular sequence analysis, aiming to explore the “murderer” who killed the massive culture-fi sh.

2 MATERIAL AND METHOD

2.1 Phytoplankton sampling

Seawater samples were collected on May 26, 2019, during the bloom in Pingtan coastal area, Fujian Province (26°22′ 39″ N, 119°52′ 42″ E) (Fig.1, station S1). Two strains of the dominated unknown naked dinofl agellate (PT-A and PT-B) were established by isolating single cell from the bloomed seawater samples. The cultures were maintained at 20°C in L1 medium (Guillard and Hargraves, 1993) at a salinity of 30, under 50 μmol photons/(m2·s) irradiance and a 12-h:12-h light:dark cycle. The cultures were deposited in the Research Center for Harmful Algae and Marine Biology, Jinan University, Guangzhou, China.

2.2 Light microscopy (LM)

Both live fi eld samples and lab cultures were observed under Olympus BX61 microscope (Olympus, Tokyo, Japan). Photographs were captured using the microscope equipped with a QImaging Retiga 4000R digital camera (QImaging, Surrey, BC, Canada). Cell nuclei and chloroplasts were visualized by epifl uorescence microscopy. For epifl uorescence micrographs, 1-mL portions of fi eld samples were fi xed with SYBR Safe DNA (Thermo Fisher, Waltham, MA, USA), and photographed with 360-370 nm excitation and 420-460 nm emission fi lters.

2.3 Cell measurements

Lengths, widths, and the degree of girdle displacement of 88 randomly selected cultured cells were measured using Image-Pro Plus 6.0 image acquisition and analysis software (QImaging) on a PC coupled to an Olympus BX61 inverted light microscope.

2.4 Scanning electron microscopy (SEM)

For SEM observations, 5-mL of each bloom sample was fi xed in a fi nal concentration of 2.5% glutaraldehyde for 12 h at 4°C. The fi xed culture cells were then fi ltered onto a Whatman fi lter (8 μm) and rinsed in deionized water for 10 min. Samples were dehydrated using a 10%, 30%, 50%, 70%, and 90% ethanol series, 20 min at each change, followed by three changes for 10 min each in 100% ethanol. The samples were subsequently critical-point dried in liquid CO2using a Leica EM CPD300 critical point dryer (Leica Microsystems, Mannheim, Germany). After gold-palladium coating, mounted samples were observed under an Ultra 55 fi eld emission scanning electron microscope (Zeiss, Jena, Germany).

2.5 PCR amplifi cation of LSU and ITS DNA regions

For fi eld samples, single cell was isolated from the seawater and transferred into 200-μL PCR tubes for PCR amplifi cation. For lab cultures, 2-mL of the cell culture was collected by centrifugation, and total genomic DNA was extracted using a TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. The D1-D3 region of the LSU of rDNA (～850 base pairs) was amplifi ed using primers D1R (Scholin et al., 1994) and D3Ca (Lenaers et al., 1989), and the ITS region was amplifi ed with primers ITS1 and ITS4 (Stern et al., 2012). PCR amplifi cations were carried out in a PTC-200 Peltier thermal cycler (MJ Research, San Francisco, CA, USA). Cycling conditions for amplifi cation of the LSU were 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min 30 s, and 72°C for 1 min, with a fi nal extension at 72°C for 10 min. The protocol for ITS amplifi cation was as follows: 94°C for 3 min, followed by 34 cycles of 94°C for 30 s, 47°C for 30 s, and 72°C for 45 s, with a fi nal extension at 72°C for 7 min. The PCR products were purifi ed and sent to Beijing Genomics Institute (Guangzhou, China) for sequencing.

2.6 Sequence alignment and phylogenetic analyses

The generated sequences were aligned in ClustalX (Thompson et al., 1997) with other representative Kareniaceae downloaded from GenBank, followed by manual refi nements. The general accepted maximum likelihood and Bayesian inference methods were carried out to analyze the phylogenetic relationship among the relevant species, using Gymnodinium catenatum and Heterosigma akashiwo as outgroup. The maximum likelihood analysis was carried out in MEGA version 6.0 (Tamura et al., 2013), and Bayesian inference was performed in MrBayes 3.1.2 after determination of the best-fi tting model (Ronquist and Huelsenbeck, 2003). Statistical support for branches in the ML and BI trees was assessed using 1 000 bootstrap iterations.

3 RESULT

3.1 Morphology and characterization of the unknown causative dinofl agellate

3.1.1 LM

Cells appeared globular or oval in shape (Fig.2a & b), with a mean length of 20.34±1.54 μm ( n=88), a mean width of 16.02±1.3 μm ( n=88), and a length:width ratio of 1.27±0.06 ( n=88). The straight apical groove extended from the dorsal apex to the ventral epicone, and located at the right side of the sulcal axis (Fig.2c). In the ventral view, the sulcus invaded the epicone slightly as a small fi nger-like extension (Fig.2d). When meeting the cingulum, the sulcus turned to left and formed curved knot on the left lobe of the hypocone (Fig.2e). The cingulum was relatively wide, median, and encircled the cell equatorially (Fig.2f).The chloroplasts were yellowgreen to yellowish brown, spherical in shape, and irregularly distributed in cells (Fig.2g & h). The large spherical nucleus was located centrally to posteriorly (Fig.2i). Cells moved smoothly and quickly in a rotating manner.

Fig.2 Light and epifl uorescence micrographs of Karenia digitata

Fig.3 Scanning electron micrographs of Karenia digitata

3.1.2 SEM

Observed under SEM, the epicone and hypocone were both hemispherical and were approximately equally sized (Fig.3a). The sulcus extended from just above the proximal end of the cingulum and elongated nearly to the antapex end. The linear apical groove was distinct under SEM, and located at the right of the sulcal axis. The cingulum displacement was approximately 25% of the total cell length (Fig.3a). The sulcus intruded onto the epicone as a fi nger-like projection (Fig.3a & b). The hypocone was conical, and did not notch by the sulcus at the dorsal side (Fig.3c). The apical groove was narrow and straight, extending about one-third of the length to the dorsal side of the epicone (Fig.3c & d). The apical groove was narrow in center, and became wide at the two ends (Fig.3e). Cell surfaces had irregularly distributed pores, mainly on the epicone (Fig.3f).

3.1.3 Morphological characterization

Based on the observation under LM and SEM, and the comparison with other morphologically similar species (Table 1), the unknown naked dinofl agellate was featured by a straight apical groove on the epicone, an obvious fi nger-like extension of the sulcus into the epicone, and a sulcal curvature on the left lobe of hypcone. The morphological characteristics of the unknown dinofl agellate were exactly in accordance with the typical features of K. digitata described in Yang et al. (2000); therefore, we identifi ed the unknown causative species as K. digitata.

3.2 Phylogeny of Karenia digitata

3.2.1 rDNA sequences

LSU sequences of four single-cell PCR products (PT01, PT02, PT03, and PT04) and two culture strains of the causative species K. digitata (PT-A and PT-B) were obtained and deposited in GenBank under accession numbers MN134472 to MN134476, and MN134478. No molecular sequence of K. digitata was provided by Yang et al. (2000) when this species was fi rst reported in 2000. In 2011, Lee et al. (2011) released the LSU and ITS rDNA sequences of a species isolated in Hong Kong, China, which they identifi ed as K. digitata (stain No. KD01). The LSU sequences of Fujian cultures in the present study were nearly identical to that of K. digitata reported in Lee et al. (2011), with only one difference erence observed among the total 704 base pairs.

Table 1 Morphological comparison of Karenia digitata with related species

ITS sequences of four single-cell PCR products (PT-05, PT-06, PT-07, and PT-08) and two culture strains of the causative species K. digitata (PT-A and PT-B) were obtained and deposited in GenBank under accession numbers MN133930 to MN133934, and MN309841. The ITS sequences of the four single-cell PCR products (615 base pairs) were identical to that of the KD01 strain of K. digitata reported by Lee et al. (2011), and the two cultures (PT-A and PT-B) difference ered from the strain KD01 by 2 bases. Therefore, the LSU rDNA and ITS rDNA sequences also proved the fact that the causative dinofl agellate was K. digitata.

3.2.2 Phylogenetic analysis

In the phylogenetic tree based on LSU rDNA sequences, K. digitata isolated in the present study were clustered with K. digitata KD01, and formed a single clade with bootstrap/posterior probability values of 96%/0.95 (Fig.4). The clade of K. digitata was most closely related to Karl. austra e l and Karl. armiger, and the three species clustered into a single branch with bootstrap/posterior probability values of 95%/0.99 (Fig.4). The phylogenetic tree constructed on ITS rDNA sequences outputted the similar results as LSU rDNA tree, and the Pingtan strains of K. digitata and Hong Kong strain (KD01) were conspecifi c, closely clustering to Karl. austral e and Karl. armiger (Fig.5).

Fig.4 Maximum-likelihood phylogenetic tree of LSU sequences of Karenia digitata and closely related species

In the tree inferred from LSU rDNA, the 10 Karenia species ( K. longicanali s, K. umbella, K. papilionacea, K. asterichroma, K. bidigitata, K. brevis, K. mikimotoi, K. cristata, K. brevisulcat a, and K. selliformis) formed a clearly single branch (with bootstrap/posterior probability values of 100%/1), while the Pingtan K. digitata as well as Hong Kong K. digitata (KD01) was strongly clustered with Karlodinium species ( Karl. australe, Karl. armiger, Karl. zhouanum, Karl. venefi cum, and Karl. micrum) with bootstrap/posterior probability values of 76%/0.99. The ITS tree also revealed the similar results with LSU rDNA that species belonging to Karenia genus formed a clade (86%/1), while K. digitata and some Karlodinium species clustered into another clade (82%/0.95). The results suggested that K. digitata was phylogenetically belonged to the genus Karlodinium rather than genus Karenia.

3.3 Revison of Karenia digitata as Karlodinium digitatum

Based on the morphological characteristics, the naked causative species was identifi ed as K. digitata. According to the phylogenetic data, this species was classifi ed to genus Karlodinium. Luo et al. (2018) performed a re-taxonomy of “ K. digitata” (strain HK1) and emended it to Karlodinium digitatum based on the LSU rDNA and ITS sequences. Although maybe based on a culture of Karl. decipiens, the combination of Karl. digitatum Gu, Chan & Lu in Luo et al. (2018) is valid and this is the correct name of the original organism K. digitata . Therefore, the naked causative dinofl agellate in Pingtan coastal areas of Fujian Province, on May 24-29, 2019, was fi nally identifi ed as Karl. digitatum.

4 DISCUSSION

Fig.5 Maximum-likelihood phylogenetic tree of ITS sequences of Karenia digitata and closely related species

Karenia digitata was fi rst described and placed in the genus Karenia because of its straight apical groove and the fi nger-like extension of the sulcus into the epicone (Yang et al., 2000). However, K. digitata more closely resembles Karlodinium species than those of Karenia in regards to body size and other morphological features (Table 1). The absence of a ventral pore was originally used to distinguish K. digitata from members of the genus Karlodinium, although species lacking a ventral pore, such as Karl. ballantinum (de Salas et al., 2008) and Karl. zhouanum (Luo et al., 2018), have subsequently been found to belong in Karlodinium. The nucleus of K. digitata is large and round, and is sub-centrally located on the posterior and left side of the hypocone; this nuclear location is difference erent from that of Karl. ballantinum (centrally located on the dorsal cell surface) and Karl. zhouanum (located in the lower epicone).

The sulcus extension into the epicone has also been observed in other Karenia and Karlodinium species. In most Karenia species, the sulcus extension invades the epicone as a small closed or open-ended triangular extension (Yang et al., 2000, Figures 18-27; Haywood et al., 2004, Figure 6), and only one species, K. umbella, has a fi nger-like sulcus extension like that of K. digitata (de Salas et al., 2004). Karenia umbella can be distinguished from K. digitata by its large cell size and the six or eight radial furrows on the epicone that are visible by SEM (Wang et al. (2018) concluded that K. umbella and K. longicanalis are the same species). Sulci in most species of Karlodinium extend into the epicone, and some, such as Karl. armiger, Karl. australe, Karl. conicum, Karl. corrugatum, and Karl. gentienii have a fi nger-like shape; however, the species with a fi nger-like sulcus intrusion all have an apparent ventral pore that allows them to be easily discriminated from K. digitata.

When K. digitata was fi rst reported, DNA sequences of the holotype were not available. Lee et al. (2011) published the LSU and ITS sequence of culture cells of K. digitata isolated from algal bloom samples collected in Silver Mine Bay during March 23-26, 2009 (The identifi cation of the causative agent as K. digitata was confi rmed morphologically by oき cials of the Agricultural, Fisheries and Conservation Department of the Hong Kong SAR Government). In 2018, Luo et al. (2018) published LSU and ITS sequences of bloom samples they collected and freeze dried during the bloom event in 1998, which they identifi ed as “ K. digitata” and emended to Karl. digitatum. The ML and BI phylogenetic analyses of LSU and ITS sequences in the present study suggested that the Pingtan strains of K. digitata are conspecifi c with the Hong Kong KD01 strain of Lee et al. (2010), and they clustered with species of Karlodinium in the phylogenetic trees; while the “ K. digitata” (HK1) reported by Luo et al. (2018) was very similar to Karlodinium decipiens, clustering with the latter in the LSU-based tree with bootstrap/posterior probability values of 100%/1.0, and formed a subclade with Takayama. In their discussion, Luo et al. (2018) suggested that LSU sequences may be too conserved to difference erentiate this group of Kareniaceae species, and this is the reason why “ K. digitata” and Karl. decipiens have identical LSU sequences even though they are signifi cantly distinct in morphology. Because there was no morphological description of “ K. digitata” (HK1) when they was pressed by Luo et al. (2018), we proposed that the sequences cannot be confi dently assigned to the bloom-causative organism K. digitata in 1998, but more likely is a strain of Karl. decipiens. The molecular sequences representing Karl. digitatum, however, are those of Lee et al. (2011) and those published in the present study.

In the present study, the causative naked species in Pingtan bloom was emended as Karl. digitatum from the original K. digitata. The genera Karenia and Karlodinium both have a straight apical groove on epicone. Morphologically, K arenia species are more dorsoventrally fl attened than Karlodinium species (Haywood et al., 2004). A key characteristics distinguishing Karlodinium from Karenia is the presence of a ventral pore in Karlodinium (Bergholtz et al., 2005, de Salas et al., 2008). However, the increasing discoveries of species, which lack a ventral pore but are genetically consistent with Karlodinium, e.g. Karl. zhouanum, and Karl. digitatum in the present study, raise the issue of what remains to difference erentiate Karlodinium from Karenia.

Karenia mikimotoi was the most common harmful bloom species in Fujian coastal areas, China (Xu et al., 2010). K. mikimotoi is much bigger and more fl attened than Karl. digitatum in body appearance. The hypocone of K. mikimotoi was sliced to bilobeshaped by the sulcus, while that of Karl. digitatum was rounded to semispherical (Yang et al., 2000; Haywood et al., 2004). The naked cell of Karenia species is easy to be deformed after fi xation when the cells are checked under LM, and this may lead to the misidentifi cation of causative species. It is uncertain whether the actual causative species of “ Karenia” was K. mikimotoi in the past years in Fujian coast.

In 1998, the Karl. digitatum (basionym: K. digitata) bloom invaded 22 of Hong Kong’s 26 coastal fi sh farms, killed 90% of caged fi sh, and caused losses of approximately HK$250 million (US$32 million) in Hong Kong, China (Yang and Hodgkiss, 1999, 2004). This species apparently fi rst appeared in Hong Kong in 1989 and then again in 1995, but it did not form a bloom until 1998. Karl. digitatum is suspected to have bloomed along the coasts of western Japan in the summers of 1995 and 1996 and in November 1997. Various fi sh species, such as red sea bream ( Pagrus major), bastard halibut ( Paralichthys olivaceus), sea bass ( Lateolabrax japonicus), fi le fi sh ( Acanthurus xanthopterus), yellow tail ( Seriola quinqueradiata), and horse mackerel ( Trachurus japonicus), were reportedly killed by this naked dinofl agellate (Landsberg, 2002). Moreover, cultured seaweeds ( Porphyra tenera) were afference ected by the bloom in Japan and exhibited irregular cell growth, which suggests that this alga is also harmful to marine plants. Strangely and interestingly, no further studies of this species were conducted until 2009 by Lee et al. (2011), and very few data have been collected worldwide on this harmful species until now.

During the bloom in coastal waters of Fujian, China, in 2019, P. donghaiense and Karl. digitatum were the most abundant species. P. donghaiense has formed high biomass in the East China Sea since 1998; however, it is neither a toxin producer nor a species associated with fi sh kills (Lu et al., 2014). As mentioned above, huge mortality of fi sh has been caused by Karl. digitatum in Hong Kong and Japan, we supposed that the “murderer” who killed the massive culture-fi sh in the bloom during May 24-29, 2019 in Pingtan coastal areas of Fujian province was Karl. digitatum.

A notable feature of Karl. digitatum collected from Pingtan is the many irregularly placed pores on the cell surface that are visible under SEM (Fig.3d). This feature was not mentioned in Yang et al. (2000), however, de Salas et al. (2005) found a row of small processes on the hypocone of K. digitata described in Yang et al. (2000) (Figures 10-12). Similar structure, trichocysts pores were observed in Karl. armiger (Bergholtz et al., 2006), a toxic species which can producd karmitoxin and kill copepod (Rasmussen et al., 2017). We hypothesis that the pores distributed on the cell surface of K arl. digitat um are those of trichocysts and are related to toxin production.

5 CONCLUSION

In this study, the second causative organism of the bloom in Fujian coastal waters, China, in 2019 was isolated and identifi ed. On the basis of its morphological characteristics and molecular sequences, the species was identifi ed as Karl. digitat um and found to be conspecifi c with K. digitata which bloomed in the coastal waters of western Japan and Hong Kong in 1998. We proposed that the massive mortality of fi sh in this bloom was induced by the toxic species Karl. digitat um.

6 DATA AVAILABILITY STATEMENT

The data that support the fi ndings of this study are available from the corresponding author upon request.

7 ACKNOWLEDGMENT

Special appreciation is expressed to Prof. Moestrup of the University of Copenhagen for helpful discussions regarding the species identifi cation. We also sincerely thank Dr. ZHEN Yu of the Ocean University of China for his assistance with the molecular data analysis.

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