The morphological and molecular detection for the presence of toxic Cylindrospermopsis (Nostocales, Cyanobacteria) in Beijing city, China*

XIE Jinlin (谢金林) , YU Gongliang (虞功亮) , XU Xudong (徐旭东) ,LI Shouchun (李守淳) , , LI Renhui (李仁辉) ,

1 College of Life Sciences, Jiangxi Normal University, Nanchang 330022, China

2 Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

1 INTRODUCTION

Cyanobacterial blooms have been reported to occur in freshwater bodies worldwide, and the occurrence of these blooms is becoming more frequent due to anthropogenic eutrophication and global warming(Paerl and Husiman, 2009; Bonilla et al., 2012; Sinha et al., 2012; Paerl and Otten, 2013). It was estimated that over 40 cyanobacterial genera were responsible for forming blooms, and subsequently likely to produce a wide range of harmful secondary metabolites, mainly including cyanotoxins and tasteodor substances (Paerl et al., 2015). Among these bloom-forming cyanobacteria,Cylindrospermopsis raciborskii(Woloszyńska) Seenayya et Subba Raju,the type species ofCylindrospermopsisgenus, is a solitary heterocystous cyanobacterium that was initially found from the lakes of Java (Indonesia) in 1912 (Komárek and Kling, 1991). This species has been gradually recognized to poses a significant threat to ecological and human health because of its formation of blooms and production of toxic secondary metabolites such as hepatotoxic cylindrospemopsin and deoxy-cylindrospermopsin,and neurotoxic saxitoxins (Lagos et al., 1999; Saker and Eaglesham, 1999). Its occurrence was initially regarded to be limited in tropical environments, but over the past several decades its prevalence further into temperate regions, including northern Europe,New Zealand and the northern states of the USA, has rapidly increased (Hong et al., 2006; Burger et al.,2007; Kouzminov and Wood, 2007; Wiedner et al.,2007; Haande et al., 2008). The strategies adopted by this species to overgrow and out-compete other species has been summarized as its ecophysiological plasticity including its tolerance of a wide range of temperatures and light intensities, high phosphate affinity and storage capacity, nitrogen fixation or using dissolved inorganic nitrogen as nitrogen availability sources fluctuate (Burford et al., 2016),and these winning strategies may greatly contribute to its global expansion.

Table 1 Characteristics of the six sites studied in Beijing

The planktonic genusRaphidiopsis, withR.curvataas the type species, is characterized by presence of akinetes (spores) but absence of heterocysts throughout the life cycle (Fritsch et al., 1929).Raphidiopsisis known to be morphologically similar toCylindrospermopsis, especially in the cases where heterocytes and akinetes are not formed inCylindrospermopsisspecies (Li et al., 2001; Moustaka-Gouni et al., 2009; Alster et al., 2010). It has been also found that bothCylindrospermopsisandRaphidiopsisgenera shared the similar ecological niches, because they both often co-occur in the same water (Moustaka-Gouni et al., 2009). Further, the very highly genetic homologies were found betweenCylindrospermopsisandRaphidiopsisgenera, with more than 99% in 16S rRNA gene sequence and even more than 90%homology at genomic level (Stucken et al., 2009).Thus, the termCylindrospermopsis/Raphidiopsisgroup has been used to represent their close association in many aspects.

Reports about occurrence of the blooms formed byCylindrospermopsis/Raphidiopsisspecies and cylindrospermopsins in China have been recently increased, but mainly restricted in the south (Jiang et al., 2014; Lei et al., 2014). To obtain more information about distribution and expansion ofCylindrospermopsisin Chinese regions, surveys on waters along subtropical into temperate regions in China were performed during past years. In this study,we presented the findings about existence ofCylindrospermopsis/Raphidiopsisin Beijing city, and results based on morphological and molecular approaches clearly demonstrated thatCylindrospermopsishas quickly invaded into the north of China and the potential production of Cylindrospermopsin by this invasive species implicated water safety problem in this important huge city.

2 MATERIAL AND METHOD

2.1 Sampling sites and sample collection

Beijing is located at longitude 115.7°–117.4°E,latitude 39.4°–41.6°N, and its climate is the typical north temperate with semi-humid continental monsoon. A sampling was performed on September 18, 2015. As shown in Fig.1, six sampling sites from different lakes/ponds were selected, and each sites were characterized as listed in Table 1. 500 mL of surface water samples were collected from the shoreline of each lakes, and all samples were kept in a cooler and brought back to the lab for further investigation.

2.2 Measures for chlorophyll a (Chl- a), total nitrogen (TN) and total phosphorus (TP)

Chl-aconcentrations were determined following the method of Jeff rey and Humphrey (1975). Water samples filtered through Whatman GF/C fiberglass membranes (NJ, USA), and add 90% ethanol into the filtered GF/C to extract the pigments. The extracts were then sonicated and kept at 4°C for 24 h to ensure all pigments to fully extracted and eluted into the ethanol. Then the extracts were centrifuged at 5 000×g(Eppendorf, Germany) for 20 min and absorptances were measured at 750, 663, 645 and 630 nm. Chl-ɑconcentration was calculated according to the formula as:

Fig.1 Location of six sampling sites

whereCis Chl-aconcentration (μg/L),V1is the volume of ethanol (mL),Vis the volume of water samples for filtration (L).

Total phosphorus (TP) was determined by the spectrophotometric determination of ammonium molybdate, and determination of total nitrogen (TN)by Ultraviolet Spectrophotometry with alkaline potassium persulfate digestion (Wu et al., 2006).

2.3 Morphological examination

The morphological features of water samples were observed under a light microscope with Olympus BX-4l (Olympus, Japan), and the micrographs were captured by the microscope with DS-Ri1 digital camera (Nikon, Japan).

2.4 PCR, cloning and sequencing

Water samples (1.5 mL) in Eppendorf tube were centrifuged (10 000×g, 5 min) and the supernatantremoved by the sterile pipette, and 1 μL of the pellet was directly used for the PCR. The primer paircyl-16sf56/cyl-16sr955with specificity targetingC.raciborskii/Raphidiopsiswas used to amplify the 16S rRNA gene fragment. Primers specific for current known CYN-producing species werecyrJf/cyrJr(Table 2). PCR was performed in a PTC-100 thermal cycler (MJ Research, USA). PCR mix was prepared in 50 μL volume containing 5 μL of 10×PCR buff er(TaKaRa, Japan), 10 nmol of each deoxynucleotide triphosphate, 10 pmol of each primer, 1 U of LA Taq(TaKaRa), and 1 μL intact cells as DNA templates.The cycling conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C 30 s, 58°C 30 s,72°C 30 s, and a final extension of 72°C 5 min; and a 4°C hold. PCR amplicons were visualized with 1%agarose gel electrophoresis with ethidium bromide staining under UV illumination.

Table 2 The primers and product sizes in this study

Table 3 The concentrations of nutrient (TN, TP) and Chlorophyll a in six sampling sites

The PCR amplicon was ligated into pMD18-T vector (TaKaRa, Japan) that was used to transformEscherichiacoilDH5ɑ competent cells. The plasmid containing the inserted DNA fragment was purified using a PCR purification kit (Generay, China)according to the manufacturer’s instructions. Two positive bacterial clones were randomly picked per clone library, and then sequencing was performed using an ABI 3730 automated sequencer (Applied Biosystems) in both directions. All available nucleotide sequences have been deposited in the GenBank database under accession numbers KX955225-KX955233 and KY234284-KY234286.

2.5 Phylogeneticanalysis

The obtained sequences of 16S rRNA andcyrJgenes in this study were used to perform the phylogenetic analyses along with the previously published sequences in the GenBank. Multiple sequence alignments were undertaken using MEGA 6(Tamura et al., 2013), manually checked for the correction, and assessed for the best substitution model for DNA sequence evolution through the calculation by akaike information criterion (AIC)using ModelTest 3.06 (Posada and Crandall, 1998).For 16S rRNA sequences, the multiple-aligned data using the neighbor-joining (NJ) algorithmic in MEGA6 program package (Tamura and Dudley,2013), was performed with 1 000 bootstrap analysis under Kimura’s two-parameter. The maximumlikelihood (ML) trees were constructed in PAUP 4.0v10b (Swoff ord, 2003), and performed with 100 bootstrap analysis based on the parameters[Nreps=100 Keepall=yes Brlens=yes], [PAUP command lines for each criterion Lset Base=(0.269 7 0.208 9 0.325 6) Nst=6 Rmat=(0.658 3 2.412 4 0.591 2 0.468 0 5.081 3) Rates=gamma Shape=0.661 4 Pinvar=0.565 0] under a best-fit model (GTR+I+G).Bayesian analyses were performed in MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001), and parameters in MrBayes were set to five million generations and 50 000 trees, sampled every 100th generation, using the GTR+I+G model of DNA substitution, Nst=6, rates=invgamma, Burnin=10 000 and the default random tree option was set to begin the analysis. ForcyrJgenes sequences, the multiplealigned data using the neighbor-joining (NJ)algorithmic Kimura’s two-parameter was implemented within MEGA6 program package(Tamura et al., 2013). The final phylogenetic tree was drawn with Adobe Photoshop CS6.

3 RESULT

3.1 Water quality parameters

The TN, TP and Chl-aconcentrations of the examined water samples were shown in Table 3. TN concentrations of all samples were above 1.2 mg/L,varied among the lakes, with the highest concentration(4.4 mg/L) measured in Qingnian Lake. TP concentrations at all sites ranged from 0.07 to 0.24 mg/L. TN/TP ratios were in the range from 7.4 to 61, and Chl-aconcentrations varied from 11.3 to 68.8 μg/L.

3.2 The morphological identification of Cylindrospermopsis raciborskii

Straight and spiral morphotypes ofC.raciborskiiwere identified, but the coiled one was not found.TypicalRaphidiopisisspecies were not observed(Fig.2). The morphotype with straight trichomes was found in four samples and the spiral one was found in Qingnian Lake. Trichomes were free-floating and solitary, and apical cells were conically narrowed and rounded. Terminal heterocysts were located at one or both of ends of the trichome with drop-like and rounded-pointed ends. Akinetes were cylindrical to oval, developing near the ends of trichomes, adjacent to the terminal heterocysts or slightly distant(separated by one to several vegetative cells) from heterocysts, usually solitary, less frequently up to 3 in a row.

Fig.2 The morphological characteristic of Cylindrospermopsis raciborskii

3.3 The PCR amplification

Amplification by primers forC.raciborskii/Raphidiopsis-specific 16S rRNA gene and CYN synthetase genecyrJfrom these samples were shown in Fig.3. Using the culture ofC.raciborskiiCHAB3438 as the reference strain, the result shows that five samples may containC.raciborskii/Raphidiopsiscells and three of them with positive amplification of thecyrJgene.

3.4 The phylogenetic tree of cyanobacteria based on 16S rRNA gene sequences

We obtained nine sequences of 16S rRNA gene from this study. Sequence alignment produced a matrix including these nine sequences and those from cyanobacterial strains deposited in GenBank including 7Raphidiopsisstrains, 19Cylindrospermopsisstrains and 7 other different cyanobacterial strains.

The phylogenetic trees based on the above 16S rRNA gene sequences using the three method as the Neighborjoining (NJ), Maximum likelihood (ML) and Bayesian inference (BI) led to the similar topological structures.As shown in Fig.4, there are three clades: a, b and c. The nine sequences obtained in this study were distributed in clades a and b. The all sequences in a clade belonged toCylindrospermopsisstrains and those in b as a mixture ofRaphidiopsisandCylindrospermopsisspp. strains,indicating high similarity of 16S rRNA gene sequences betweenCylindrospermopsisandRaphdiopsisspecies with addition of Chinese strains.

Fig.3 PCR products of 16S rRNA and cyrJ genes displayed on a 1% agarose gel

3.5 The phylogenetic tree of cyanobacteria based on cyrJ gene

Fig.4 Neighbor-joining (NJ) phylogenetic tree based on the 16S rRNA gene sequences containing 7 strains Raphidiopsis,19 strains Cylindrospermopsis, and 7 strains from different cyanobacterial species (obtained from NCBI) and 9 cyanobacteria clones (this study)

ThreecyrJgene sequences were obtained from this study. A phylogenetic tree was constructed based oncyrJsequences from these three sequences andcyrJsequences deposited in GenBank from 6Cylindrospermopsisstrains, 3Raphidiopsisstrains and 9 other cyanobacterial ones. The tree, as shown in Fig.5, was built using the neighbor-joining (NJ)algorithmic Kimura’s two-parameter as implemented within MEGA6 program package (Tamura et al.,2013). The tree showed that allcyrJsequences obtained in this study were belonging to theCylindrospermopsis/Raphidiopsisgroup with high bootstrap supports, suggesting thatCylindrospermopsisorRaphidiopsisspecies in these samples have potential ability to produce hepatotoxic cylindrospermopsins.

Fig.5 Neighbor-joining (NJ) phylogenetic tree based on the cyrJ gene sequences from 3 strains Raphidiopsis, 6 strains Cylindrospermopsis, and 9 strains from different cyanobacterial species (obtained from NCBI) and 3 cyanobacteria clones (this study)

4 DISCUSSION

Recently, more and more studies onC.raciborskiihave been worldwide performed sinceC.raciborskiiis able to form water blooms and produce cylindrospermopsin and neurotoxins. These studies mainly targeted on detection ofC.raciborskiiblooms,the geographical distribution ofC.raciborskii, types of cyanotoxins produced by this species and toxicity of cylindrospermopsins on aquatic animals and mammals (St. Amand, 2002; Griffiths and Saker,2003; Haande et al., 2008). In contrast to other global regions / countries, only a few investigations on the occurrence, toxin detection and molecular characterization ofCylindrospermopsis/Raphidiopsisgroup have been done from Chinese waters. Li et al.(2001) reported the production of deoxycylindrospermopsin and cylindrospermopsin from a strain ofRaphidiopsiscurvataHB1 isolated from a fish pond in Wuhan, China, representing the first study on cylindrospermopsin ofCylindrospermopsis/Raphidiopsisgroup in China. Wu et al. (2011) used the 21 strains, from species ofC.raciborskii,R.curvataandR.mediteriannisolated from subtropical southern provinces of Hubei, Yunnan, Fujian and Guangdong, to perform phylogenetic analyses based on multi-gene sequences for molecular differentiation betweenCylindrospermopsisandRaphidiopsisgenera. Further, Lei et al. (2014) documented the occurrence and dominance ofC.raciborskiiand production of cylindrospermopsin in 25 urban reservoirs used for drinking water supply in Dongguan city, Guangdong Province, and they even detected dissolved cylindrospermopsin in more than half of these reservoirs with up to 8.25 μg/L concentrations.Apparently,C.raciborskiihas been found in tropical to subtropical regions of China, even in the transitional zone between subtropical to temperate climate, such as in Jiangsu and Shandong provinces (data not shown). It is challenging to determine if the detection ofC.raciborskiiin new regions has occurred due to invasions or as a result of an increase in its abundance caused by changes in environmental conditions (e.g.,Wood et al., 2014). Previous studies have suggested that phylo-biogeographic patterns can be used to reflect global origin and spreading routes ofC.raciborskii(Dyble et al., 2002; Neilan et al., 2003;Gugger et al., 2005; Moreira et al., 2015). However,recent works have shown a more nuanced story when a greater number of strains from more regions are included (Cirés et al., 2014; Antunes et al., 2015).Therefore, the confirmation for the spreading ofC.raciborskiiin China may not be able to be determined by phylo-biogeographic analysis. Based on the absence of this species in previous phycological studies in this region we suggestion the most likely scenario is that it is a new incursion. The occurrence in the transitional zone in China is of particular concern with very characteristic signs of invasion and expansion ofC.raciborskii. Therefore, more investigations on the occurrence ofC.raciborskiiand its impact in further northern regions in China are much needed to understand the invasive feature ofC.raciborskiiin China and physio-ecological mechanisms for its expansion.

In this study, we examined and confirmed the occurrence of cylindropsermopsin-producingCylindrospermopsis/Raphdiopsisinurban lakes of Beijing city, China, and this result represented the most northern line ofCylindrospermopsisdistribution so far reported in China, and therefore contributes to the knowledge and methods in morphological identification ofCylindrospermopsis, and further in molecular detection ofCylindrospermopsis/Raphdiopsisand their potential production of cylindrospermopsin. The future studies will mainly include the isolation ofCylindrospermopsis/Raphdiopsisstrains, detection of their cylindrospermopsin production and monitoring dynamics ofCylindrospermopsis/Raphdiopsisand cylindrospermopsin in Beijing waters.

In addition to the description about the ecophysiological plasticity ofC.raciborskii, Padisák(1997) summarized that the reasons forC.raciborskiisucceeding in the world’s lakes might be attributed to multiple factors such as good floating ability, superior shade tolerance, high affinity ammonia uptake and resistance to grazing. Therefore, climate change with global warming tendency and ecophysiological advantage ofC.raciborskiihave been regarded as possible mechanisms for the gradual expansion and dominance ofC.raciborskiiinto northern regions in China. According to the data from Intergovernmental Panel on Climate Change (IPCC), the average temperatures in the northern and southern hemispheres during the 20th century have risen 0.52°C and 0.7°C,respectively (IPCC, 2001). Further, the annual average temperature in Beijing was the rise of 0.5°C per 10 years (Zhu et al., 2012), much faster than the average in global range. Result of this study may explain that this temperature rise in Beijing already provided the condition with a suitable temperature forC.raciborskiito grow and proliferate. In the present study, water temperatures in the six sites in mid-September, with average 25.6°C, were similar to those in waters of typical subtropical regions in China.In Chinese subtropical areas, our previous surveys showed thatC.raciborskiisamples were always found in autumn season, after the summer months with frequent occurrence ofMicrocystisdominated blooms at higher temperatures. Several investigations have indicated that theMicrocystisblooms have been previously reported in urban lakes in Beijing during summer season (Tu et al., 2004; Du et al., 2005).Therefore, the pattern forC.raciborskiioccurrence after summer in Beijing is also similar to the case in Chinese southern waters.

Water quality parameters, with major concern to nutrition such as TN and TP in this study, showed that the six water bodies were already eutrophicated (HLJ,TZ and LHC as super-eutrophicated) (Table 2),according to Classification References Standard of Lake Nutrition (Smith et al., 1999), and such eutrophic levels in the lakes create the proper nutrient conditions forC.raciborskii(Briand et al., 2004). Site YL did not haveC.raciborskiibut had the highest TN value as 4.398 mg/L, and this high value much exceeded the total nitrogen levels forC.raciborskiioccurrence from previous reports at different parts of the world.

The morphological and molecular similarities betweenCylindrospermopsisandRaphidiopsisgenera led to the difficulty in distinguishing them.McGregor and Fabbro (2000) proposed that theRaphidiopsis-like trichomes were considered as environmental morphotypes ofC.raciborskii.Moustaka-Gouni et al. (2009) suggested from the morphological and 16S rRNA phylogenetic analyses thatR.mediterraneafrom Lake Kastoria was the nonheterocystous stage in a complex life cycle ofCylindrospermopsis. In this study, 16S rRNA gene sequences from the positive PCR amplification at the five respective sites were divided into the different sub-clusters in each of which bothCylindrospermopsisandRaphdiopsiswere included, although the bigCylindrospermopsis/Raphdiopsiscluster was significantly shown. Molecular detection revealed that three of the five sites them were found to contain cylindrospermopsin synthesis genecyrJ. These lakes/ponds are located at the center of Beijing city, and these waters (LHC, NS, HLJ) have the potential ability to produce cylindrospermopsin. Even they are not currently used as for drinking water, but it is likely for them to contaminate other water resources including drinking waters due to the expansive and transportable paths ofCylindrospermopsis/Raphdiopsiswithin watershed network in the whole Beijing city. Therefore, it is strongly suggested that the monitoring onC.raciborskii/Raphidiopsisspecies and their production of cylindrospemopsin should be emphasized in Beijing and even more northern parts of China.

5 CONCLUSION

This study used morphological and molecular methods to detect toxicCylindrospermopsis/Raphidiopsisgroup in Beijing city. The eutrophic waters and global warming provide ideal conditions forC.raciborskiito spread, and the electrophoretic results of PCR products and the phylogenetic tree based on 16S rRNA indicated thatC.raciborskii/Raphidiopsissp. has already invaded into Beijing and existthe possibility to produce cylindrospermopsin.

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