The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 13, 2019

Molecular Analysis of the Cyanobacterial Community in Gastric Contents of Egrets with Symptoms of Steatitis



Tomoyasu Nishizawa1, *, Yasuko Neagari2, Takamasa Miura1, , Munehiko Asayama1, Koichi Murata3, Ken-Ichi Harada4, Makoto Shirai1,
1 College of Agriculture, Ibaraki University, Ibaraki 300-0393, Japan
2 Laboratory for Intellectual Fundamentals for Environmental Studies, National Institute for Environmental Studies, Ibaraki 305-8506, Japan
3 College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan;
4 Graduate School of Environmental and Human Science and Faculty of Pharmacy, Meijo University, Aichi 468-8503, Japan;

Abstract

Many deaths of wild birds that have drunk water contaminated with hepatotoxic microcystin-producing cyanobacteria have been reported. A mass death of egrets and herons with steatitis were found at the agricultural reservoir occurring cyanobacterial waterblooms. This study aimed to verify a hypothesis that the egrets and herons which died in the reservoir drink microcystin-producing cyanobacteria and microcystin involves in the cause of death as well as the symptoms of steatitis. The cyanobacterial community in gastric contents of egrets and herons that died from steatitis was assessed using cyanobacterial 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) profiling and a cyanobacterial 16S rRNA-based clone library analysis. In addition, PCR amplification of the mcyB–C region and the mcyG gene, involved in microcystin biosynthesis, was examined. The cyanobacterial community in the gastric contents of two birds showed a simplistic composition. A comparison of cyanobacterial T-RFLP profiling and cloned sequences suggested that the genus Microcystis predominated in both samples of egrets died. Although we confirmed that two egrets which died in the reservoir have taken in cyanobacterial waterblooms containing the genus Microcystis, no mcy gene was detected in both samples according to the mcy gene-based PCR analysis. This study is the first to show the profiling and traceability of a cyanobacterial community in the gastric contents of wild birds by molecular analysis. Additionally, we consider causing symptoms of steatitis in the dead egrets.

Keywords: Agricultural reservoir, cyanobacterial community, gastric content, Microcystis, microcystin biosynthesis (mcy) gene, T-RFLP profiling.


Article Information


Identifiers and Pagination:

Year: 2015
Volume: 9
First Page: 160
Last Page: 166
Publisher Id: TOMICROJ-9-160
DOI: 10.2174/1874285801509010160

Article History:

Received Date: 29/12/2014
Revision Received Date: 3/5/2015
Acceptance Date: 1/8/2015
Electronic publication date: 3/11/2015
Collection year: 2015

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© Nishizawa et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the (https://creativecommons.org/licenses/by/4.0/legalcode), which permits unrestricted, noncommercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the College of Agriculture, Ibaraki University, 3-21-1 Chuou, Ami, Ibaraki 300-0393, Japan; Tel: +81 29 888 8687; Fax: +81 29 888 8525; E-mail: nishitom@mx.ibaraki.ac.jp Present address: Biological Resource Center, National Institute of Technology and Evaluation, Tokyo 151-0066, Japan; ‡Present address: College of Human and Cultural Sciences, Aikoku Gakuen University, Chiba, 284-0005, Japan




INTRODUCTION

There has been increased concern over the effects on wildlife of toxins, particularly those associated with harmful algal blooms in water reservoirs and recreational areas. Cyanobacteria, which include the genera Anabaena,Microcystis, Nostoc, and Planktothrix, are known as producers of hepatotoxic microcystins [1Matsushima R, Yoshizawa S, Watanabe MF, et al. In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and fibroblasts Biochem Biophys Res Commun 1990; 171: 867-74.
[http://dx.doi.org/10.1016/0006-291X(90)91226-I]
, 2Sivonen K, Jones G. Cyanobacterial toxins. London: WHO, E and FN Spon 1999; pp. 41-111.], the toxicity of which is due to the inhibition of protein phosphatase 1 and 2A [3MacKintosh C, Beattie KA, Klumpp S, Cohen P, Codd GA. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants FEBS Lett 1990; 264: 187-92.
[http://dx.doi.org/10.1016/0014-5793(90)80245-E]
, 4Nishiwaki-Matsushima R, Ohta T, Nishiwaki S, et al. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR J Cancer Res Clin Oncol 1992; 118: 420-4.
[http://dx.doi.org/10.1007/BF01629424]
]. Microcystins acutely cause cytokeratin hyperphosphorylation which leads to a disruption of cytoskeletal components and cell deformation, followed by disruption of the liver architecture [5Carmichael WW. The toxins of cyanobacteria Sci Am 1994; 270: 64-72.
[http://dx.doi.org/10.1038/scientificamerican0194-78]
]. To date, the mcy gene encoding microcystin synthetase was characterized from the planktonic unicellular Microcystis [6Nishizawa T, Asayama M, Fujii K, Harada K-I, Shirai M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp J Biochem 1999; 126: 520-9.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481]
-8Tillett D, Dittmann E, Erhard M, von Döhren H, Börner T, Neilan BA. Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system Chem Biol 2000; 7: 753-64.
[http://dx.doi.org/10.1016/S1074-5521(00)00021-1]
], the filamentous Planktothrix [9Christiansen G, Fastner J, Erhard M, Börner T, Dittmann E. Microcystin biosynthesis in Planktothrix: Genes, evolution, and manipulation J Bacteriol 2003; 185: 564-72.
[http://dx.doi.org/10.1128/JB.185.2.564-572.2003]
], and the filamentous and heterocystic Anabaena [10Rouhiainen L, Vakkilainen T, Siemer S, Buikema W, Haselkorn R, Sivonen K. Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90 Appl Environ Microbiol 2004; 70: 686-92.
[http://dx.doi.org/10.1128/AEM.70.2.686-692.2004]
]. Microcystins are produced by a large multisubunit enzyme complex comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs).

The deaths of over 50 hemodialysis patients in Caruaru, Brazil, were attributed to exposure to microcystins in the dialysis solution [11Jochimsen EM, Carmichael WW, An JS, et al. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil N Engl J Med 1998; 338: 873-8.
[http://dx.doi.org/10.1056/NEJM199803263381304]
]. This type of toxin was responsible for the deaths of wild birds that drunk from a pond with cyanobacterial waterblooms in Hyogo Prefecture, Japan [12Matsunaga H, Harada K-I, Senma M, et al. Possible cause of unnatural mass death of wild birds in a pond in Nishinomiya, Japan: sudden appearance of toxic cyanobacteria Nat Toxins 1999; 7: 81-4.
[http://dx.doi.org/10.1002/(SICI)1522-7189(199903/04)7:2<81::AID-NT44>3.0.CO;2-O]
]. In the San Diego area, USA, death of Great Blue Herons (Ardea herodias) and Black-crowned Night Herons (Nycticorax nycticorax) caused steatitis, an inflammation of fat tissue, has been reported from 1979 [13Pavlat J, Beach H, Platter-Rieger FM, Faulkner M. Steatitis in herons and egrets in Southern California Proceedings International wildlife rehabilitation council 27th annual conference. 1-6.]. Also, the deaths of herons from steatitis was observed in Chesapeake Bay, USA, where toxic cyanobacterial waterblooms containing Anabaena spp. occurred [14Rattner AB, McGowan CP. Potential hazards of environmental contaminants to avifauna residing in the Chesapeake Bay estuary Waterbirds 2007; 30: 63-81.
[http://dx.doi.org/10.1675/1524-4695(2007)030[0063:PHOECT]2.0.CO;2]
]. Hypotheses concerning the cause of steatitis include consumption of food high in polyunsaturated fats and intake of the toxic waterbloom, hepatotoxic microcystin [13Pavlat J, Beach H, Platter-Rieger FM, Faulkner M. Steatitis in herons and egrets in Southern California Proceedings International wildlife rehabilitation council 27th annual conference. 1-6., 14Rattner AB, McGowan CP. Potential hazards of environmental contaminants to avifauna residing in the Chesapeake Bay estuary Waterbirds 2007; 30: 63-81.
[http://dx.doi.org/10.1675/1524-4695(2007)030[0063:PHOECT]2.0.CO;2]
]. Microcystins were detected in water samples and in tissue samples from the dead herons and suggested to be a possible cause of the death as well as the symptoms of steatitis [14Rattner AB, McGowan CP. Potential hazards of environmental contaminants to avifauna residing in the Chesapeake Bay estuary Waterbirds 2007; 30: 63-81.
[http://dx.doi.org/10.1675/1524-4695(2007)030[0063:PHOECT]2.0.CO;2]
]. In a similar report, more than 70 egrets and herons were found sick or dead at an agricultural reservoir in Kanagawa Prefecture, Japan [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
]. It was confirmed that these birds also died from steatitis. High counts of cyanobacteria were found in the reservoir, and the genera Microcystis, Raphidiopsis and Planktothrix were identified from cell and colony morphology. However, no microcystin was detected in the reservoir’s water or the livers of the dead birds [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
]. To verify that toxic cyanobacteria is responsible for the cause of death, it is necessary to reveal whether egrets and herons which died in the reservoir drunk the cyanobacterial waterblooms containing microcystin-producer.

Molecular methods such as 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) [16Liu W-T, Marsh TL, Cheng H, Forney LJ. Characterization of microbial diversity by determining terminal restriction fragment length polymorphism of genes encoding 16S rRNA Appl Environ Microbiol 1997; 63: 4516-22., 17Marsh TL. Terminal restriction fragment length polymorphism (T-RFLP): An emerging method for characterizing diversity among homologous populations of amplification products Curr Opin Microbiol 1999; 2: 323-7.
[http://dx.doi.org/10.1016/S1369-5274(99)80056-3]
] have been utilized to assess the dominant microbes in environmental samples using an automated DNA sequencer with quenching-fluorescence-labeling fragments [18Nishizawa T, Komatsuzaki M, Kaneko N, Ohta H. Archaeal diversity of upland rice field soils assessed by the terminal restriction fragment length polymorphism method combined with real time quantitative-PCR and a alone library analysis Microbes Environ 2008; 23: 237-43.
[http://dx.doi.org/10.1264/jsme2.23.237]
-20Sato Y, Hosokawa K, Fujimura R, et al. Nitrogenase activity (acetylene reduction) of an iron-oxidizing Leptospirillum strain cultured as a pioneer microbe from a recent volcanic deposit on Miyake-jima, Japan Microbes Environ 2009; 24: 291-6.
[http://dx.doi.org/10.1264/jsme2.ME09139]
]. T-RFLP profiling has become a valuable technique in microbial ecology and can be performed quickly and easily with a high resolution that allows even related strains of bacteria to be differentiated [21Davidson AN, Chee-Sanford J, Lai HY, Ho C-H, Klenzendorf JB, Kirisits MJ. Characterization of bromate-reducing bacterial isolates and their potential for drinking water treatment Water Res 2011; 45: 6051-62.
[http://dx.doi.org/10.1016/j.watres.2011.09.001]
-25Quaak FC, Kuiper I. Statistical data analysis of bacterial t-RFLP profiles in forensic soil comparisons Forensic Sci Int 2011; 210: 96-101.
[http://dx.doi.org/10.1016/j.forsciint.2011.02.005]
]. The genetic characterization of cyanobacterial strains has been performed using a bacterial 16S rRNA-based amplified rDNA restriction analysis (ARDRA) [26Lyra C, Hantula J, Vainio E, Rapala J, Rouhiainen L, Sivonen K. Characterization of cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene Arch Microbiol 1997; 168: 176-84.
[http://dx.doi.org/10.1007/s002030050485]
]. Generally, the 16S rRNA sequence or 16S-23S rRNA internal transcribed spacer sequence (16S-23S ITS) is utilized to classify Microcystis species [27van Gremberghe I, Leliaert F, Mergeay J, et al. Lack of phylogeographic structure in the freshwater cyanobacterium Microcystis aeruginosa suggests global dispersal PLoS One 2011; 6: e19561.
[http://dx.doi.org/10.1371/journal.pone.0019561]
]. Additionally, Nübel et al. [28Nübel U, Garcial-Pichel F, Muyzer G. PCR primers to amplify 16S rRNA genes from cyanobacteria Appl Environ Microbiol 1997; 63: 3327-32.] provided cyanobacterial specific primers for the selective retrieval of cyanobacterial ARDRA data from an environmental sample. The cyanobacterial primers were used for the identification, typing, and monitoring of cyanobacteria in freshwater reservoirs [29Valério E, Chambel L, Paulino S, Faria N, Pereira P, Tenreiro R. Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs Microbiology 2009; 155: 642-56.
[http://dx.doi.org/10.1099/mic.0.022848-0]
].

The objective of this study was to characterize the cyanobacterial community in the gastric contents of dead egrets. To assess the cyanobacterial community structure, cyanobacterial 16S rRNA-based T-RFLP profiling as well as a clone library analysis was performed. Moreover, to examine whether toxic cyanobacteria exist in the gastric contents, PCR amplification of the mcy gene was carried out.

MATERIALS AND METHODS

Collection of Samples and Cyanobacterial Strains

The agricultural reservoir was located in Yokosuka, Kanagawa Prefecture, Japan (35(12’ N, 139(37’ E). Many egrets and herons were died in biotope which is installed in the center of the reservoir. Deep frozen gastric contents of egrets and herons were provided by the National Institute for Environmental Studies (NIES, Tsukuba, Japan) (Table 1). Six samples of the gastric contents were stored at -80oC until DNA was extracted. Microcystis aeruginosa NIES-87 was obtained from NIES. M. aeruginosa strains B-19 and K-139 were isolated from Lake Kasumigaura in Ibaraki Prefecture, Japan [6Nishizawa T, Asayama M, Fujii K, Harada K-I, Shirai M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp J Biochem 1999; 126: 520-9.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481]
]. Strains were grown in CB medium at 30oC with continuous illumination under fluorescent (cool white) lights (35 µmol/m2/s) [30Shirai M, Matsumaru K, Ohtake A, Takamura Y, Aida T, Nakano M. Development of a solid medium for growth and isolation of axenic Microcystis strains (cyanobacteria) Appl Environ Microbiol 1989; 55: 2569-71.].

DNA Extraction and Manipulation

Total cyanobacterial DNA was isolated from cells grown to the late logarithmic phase using a previously described procedure [6Nishizawa T, Asayama M, Fujii K, Harada K-I, Shirai M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp J Biochem 1999; 126: 520-9.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481]
]. The samples of gastric content (0.5 g wet weight) cryopreserved for one year and a half were washed with TES buffer (0.05 M Tris-HC1, 0.1 M NaC1, 0.05 M EDTA; pH 8.0) and resuspended in 0.5 ml of SET buffer (0.05 M Tris-HCl, 0.05 M EDTA, 25% [wt/vol] sucrose; pH 8.0). Lysozyme treatment was performed on ice for 5 hours. Total DNA was isolated as described previously [6Nishizawa T, Asayama M, Fujii K, Harada K-I, Shirai M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp J Biochem 1999; 126: 520-9.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481]
].

Amplification of the Bacterial and Cyanobacterial 16S rRNA Genes

The bacterial 16S rRNA gene in extracted DNA (0.1 µg) was amplified using the forward primer bac10F and reverse primer bac1492R [20Sato Y, Hosokawa K, Fujimura R, et al. Nitrogenase activity (acetylene reduction) of an iron-oxidizing Leptospirillum strain cultured as a pioneer microbe from a recent volcanic deposit on Miyake-jima, Japan Microbes Environ 2009; 24: 291-6.
[http://dx.doi.org/10.1264/jsme2.ME09139]
], and PCR amplification was performed as described previously [20Sato Y, Hosokawa K, Fujimura R, et al. Nitrogenase activity (acetylene reduction) of an iron-oxidizing Leptospirillum strain cultured as a pioneer microbe from a recent volcanic deposit on Miyake-jima, Japan Microbes Environ 2009; 24: 291-6.
[http://dx.doi.org/10.1264/jsme2.ME09139]
]. The cyanobacterial 16S rRNA gene in extracted DNA (0.1 µg) was amplified using the forward primer CYA106f (5’-CGGACGGGTGAG TAACGCGTGA-3’) [28Nübel U, Garcial-Pichel F, Muyzer G. PCR primers to amplify 16S rRNA genes from cyanobacteria Appl Environ Microbiol 1997; 63: 3327-32.] and reverse primer CYA792r (5’-TCCCCTAGCTTTCGTCCC-3’) following the MiCA3 program [31Shyu C, Soule T, Bent SJ, Foster JA, Forney LJ. MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes Microb Ecol 2007; 53: 562-70.
[http://dx.doi.org/10.1007/s00248-006-9106-0]
]. The reaction was performed in a Takara PCR Thermal Cycler Personal (Takara Bio, Otsu, Japan) with Takara Ex Taq polymerase (Takara Bio) under the following conditions: 2 min at 95oC, followed by a cycle of 95oC (30 s), 54oC (45 s), and 72oC (90 s) and a final extension for 5 min at 72oC. To avoid PCR bias [32Kanagawa T. Bias and artifacts in multitemplate polymerase chain reactions (PCR) J Biosci Bioeng 2003; 96: 317-23.
[http://dx.doi.org/10.1016/S1389-1723(03)90130-7]
] and the heteroduplex-form of 16S rRNA [33Kurata S, Kanagawa T, Yamada K, et al. Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer Nucleic Acids Res 2001; 29: e34.
[http://dx.doi.org/10.1093/nar/29.6.e34]
], the number of amplification cycles was fixed at twenty-two in KS2P and KS4P samples. The cyanobacterial 16S rRNA gene PCR amplicon was observed at approximately 650-bp on the agarose gels. To construct the clone library, the amplicon of the cyanobacterial 16S rRNA gene region was ligated into the pGEM-T-easy cloning vector (Promega, Madison, WI) and ligation products were transformed into Escherichia coli DH5aMCR (Cosmo Bio, Tokyo, Japan) as described previously [18Nishizawa T, Komatsuzaki M, Kaneko N, Ohta H. Archaeal diversity of upland rice field soils assessed by the terminal restriction fragment length polymorphism method combined with real time quantitative-PCR and a alone library analysis Microbes Environ 2008; 23: 237-43.
[http://dx.doi.org/10.1264/jsme2.23.237]
]. PCR products were resolved on 1% agarose gels in Tris-Boreate-EDTA (TBE) buffer.

T-RFLP Profiling

The cyanobacterial 16S rRNA gene in the extracted DNA was amplified using CYA106f (see the above section for the nucleotide sequence) and QCYA792r (5’-CTCCCCTAGCT TTCGTCCC-3’). The 5’-end fluorescence-labeled primer [34Kurata S, Kanagawa T, Magariyama Y, et al. Reevaluation and reduction of a PCR bias caused by reannealing of templates Appl Environ Microbiol 2004; 70: 7545-9.
[http://dx.doi.org/10.1128/AEM.70.12.7545-7549.2004]
], QCYA792r, was purchased from Nippon Steel & Sumikin Eco-Tech Crop., Ltd. (Tsukuba, Japan). The PCR mixture (30 µl) was prepared by combining 0.1-0.01 µg of template-extracted DNA, 1.0 µl of 10 pmol/µl primers, Takara Ex Taq polymerase, dNTPs, and 3.0 µl of optimized 10-fold Ex buffer (Takara Bio) in a PCR thermal cycler. The PCR of the 16S rRNA gene for T-RFLP profiling was carried out under the following conditions: 1 min at 95oC, followed by 95oC (30 s), 54oC (45 s), and 65oC (90 s).

The PCR-based DNA fragment analysis by T-RFLP was performed as described previously [35Nishizawa T. Zhaorigetu, Komatsuzaki M, Kaneko N, Ohta H. Molecular characterization of fungal communities in non-tilled, cover-cropped upland rice field soils Microbes Environ 2010; 25: 204-10.
[http://dx.doi.org/10.1264/jsme2.ME10108]
]. The purified T-RF DNA was mixed with 15 µl of Hi-Di formamide and 0.1 µl of DNA standard LIZ® 500 (Applied Biosystems) for standardization. The precise lengths of terminal restriction fragments (T-RFs) from the amplified fragments were determined on PE Applied Biosystems Automated DNA Sequencer (model 3130xl) (Applied Biosystems, Foster, CA). The lengths of fluorescently labeled T-RFs were determined by comparison with internal standards using GeneMapper (version 3.7) software (Applied Biosystems). T-RFLP profiling of the cyanobacterial community structure in samples were obtained with peaks ranging from 0 to 700 bases. The definition of the T-RFs were peaks with a fluorescence threshold of more than 30.

DNA Sequencing and Computer Analysis

Nucleotide sequences were determined with 3130xl DNA sequencer (Applied Biosystems) and DNA sequencing reactions were carried out using a BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the protocol of the manufacturer. The double-stranded DNA sequences were assembled and analyzed using Genetyx (version 11.0) and Genetyx-ATSQ (version 4.0) software (Genetyx Co., Tokyo, Japan), respectively. Similarity to DNA sequences retrieved from the DDBJ/EMBL/GenBank databases using BLAST.

PCR Amplification of the mcyG Gene and Non-Coding Region

To amplify the mcyG gene, which is involved in the biosynthesis of Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) in microcystin [7Nishizawa T, Ueda A, Asayama M, et al. Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin J Biochem 2000; 127: 779-89.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022670]
], the primers 5’-McyG12AT and 3’-McyG12AT [36Noguchi T, Shinohara A, Nishizawa A, et al. Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan J Gen Appl Microbiol 2009; 55: 111-23.
[http://dx.doi.org/10.2323/jgam.55.111]
] and 0.1-0.01 µg of template-extracted DNA were used. The reaction was performed in a PCR thermal cycler. For the amplification, Takara LA Taq polymerase (Takara Bio) was used. The reaction was performed under the following conditions: 3 min at 95oC, followed by 5 cycles of 95oC (30 s), 54oC (45 s), and 72oC (90 s), and then 30 cycles of 95oC (30 s), 60oC (30 s), and 72oC (90 s) and a final extension for 3 min at 72oC. To generate the amplicon between the mcyB and mcyC genes, primers sets were used as described previously [6Nishizawa T, Asayama M, Fujii K, Harada K-I, Shirai M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp J Biochem 1999; 126: 520-9.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481]
, 37Nishizawa T, Nishizawa A, Asayama M, Harada K-I, Shirai M. Diversity within the microcystin biosynthetic gene clusters among the genus Microcystis Microbes Environ 2007; 22: 380-90.
[http://dx.doi.org/10.1264/jsme2.22.380]
]. To generate the amplicon of the non-coding region between the dnaN and uma1 genes and between the hypX and uma1 genes, primers set were used and the dnaN-uma1 region and the hypX-uma1 region were amplified as described previously [37Nishizawa T, Nishizawa A, Asayama M, Harada K-I, Shirai M. Diversity within the microcystin biosynthetic gene clusters among the genus Microcystis Microbes Environ 2007; 22: 380-90.
[http://dx.doi.org/10.1264/jsme2.22.380]
]. For positive controls of PCR amplification, microcystin-producing M. aeruginosa K-139, and non-mcy-possessing M.aeruginosa strains, B-19 (for amplification of the dnaN-uma1 region) and NIES-87 (for the hypX-uma1 region), were used. These PCR products were resolved on 1% and 1.5% agarose gels in TBE buffer.

Sequence Accession Numbers

All cloned sequences in this study were deposited in the DNA Database of Japan (DDBJ) under accession numbers AB689760 to AB689772.

RESULTS AND DISCUSSION

As the gastric contents of egrets and herons died have been cryopreserved in the NIES, molecular analysis may reveal the existence of toxic cyanobacteria. In this study, we succeeded in extracting gastric content DNA from two (KS2P and KS4P) of six deep frozen samples (Table 1). The PCR amplification for the bacterial 16S rRNA gene was confirmed in the KS2P and KS4P samples (Table 1). In the KS1P, KS3P, KS5P, and KS6P samples, there were almost no gastric contents since it became weak and was not able to have diets [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
]. In addition, quite slightly DNA extracted was fragmented finely and PCR amplification was impossible. The DNA samples, KS2P and KS4P, were used for subsequent analyses.

To examine the principle structure of the cyanobacterial community in the DNA samples of gastric content, 16S rRNA-based T-RFLP profiling was performed. A relative peak height of over 0.1 was observed in HaeIII-digested T-RFLP profiling (Fig. 1): 419.4-base (KS2P) and 419.3-base (KS4P) experimental T-RFs (T-RFsexp) were mainly detected. The cyanobacterial community in both the gastric contents of the birds had a simplistic composition. To identify the dominant T-RFexp, a cyanobacterial 16S rRNA-based clone library of gastric content was analyzed. A total of 13 clones were sequenced and taxonomic classification was carried out with the BLAST tool of the NCBI-nr database. Most of the cloned sequences from gastric content were closely related to the genus Microcystis, while clone KS2-4 was classified into the genus Anabaena (Table 2). The sequence of clone KS2-7 was identical with those of KS4-7 and KS4-18, suggesting that the two egrets have taken the same diets containing Microcystis. On the other hand, the in silico T-RF of the cyanobacterial 16S rRNA gene’s cloned sequence (T-RFseq) was also used for comparison as shown in Table 2. The dominant HaeIII-digested T-RFexps of KS2P and KS4P were mostly consistent with the HaeIII 421-base T-RFseq of those clones, respectively, except for clone KS2-4. These results indicated that Microcystis is the dominant genus in the gastric contents of the egrets died. The wild birds had been seen drinking water from the reservoir where the planktonic Microcystis-blooms were observed [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
], indicating that the two egrets died may drink the reservoir’s water.

Fig. (1)

T-RFLP profiling of the cyanobacterial 16S rRNA gene amplified as template total DNA of the gastric content of dead egrets. KS2P (A) and KS4P (B) were used for PCR amplification. HaeIII was used for digestion. The x-axis indicates the terminal restriction fragment length (bases) between 0 and 700 bases, and the y-axis represents the relative height of the peak. The highest peak was calculated as 1. A relative height of over 0.1 showed the T-RF length.



Fig. (2)

Detection of PCR amplicons by agarose gel electrophoresis. M, Marker; K-139, M. aeruginosa K-139; B-19, M. aeruginosa B-19; NIES-87, M. aeruginosa NIES-87.



Table 1

Summary of DNA extraction and PCR amplification from gastric contents.




Table 2

Detected T-RF sizes based on cyanobacterial T-RFLP profiling and in silico T-RF of the cyanobacterial 16S rRNA gene’s cloned sequence.




The deaths of wild herons in an area of Chesapeake Bay containing harmful algal blooms and toxic cyanobacteria suggested the involvement of a cyanobacterial toxin (microcystin) [14Rattner AB, McGowan CP. Potential hazards of environmental contaminants to avifauna residing in the Chesapeake Bay estuary Waterbirds 2007; 30: 63-81.
[http://dx.doi.org/10.1675/1524-4695(2007)030[0063:PHOECT]2.0.CO;2]
]. Cyanobacterial waterblooms were observed at the surface of the agricultural reservoir where egrets and herons died in Sep. and Oct. 2008, but microcystin was not detected by instrumental analysis (high-performance liquid chromatography, HPLC) from the reservoir’s water on Oct. 2008 and egrets and herons died [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
]. To date, no toxic strains of the genus Microcystis have been investigated in relation to the microcystin biosynthesis (mcy) gene. Although a highly conserved gene organization of the mcyABC and mcyDEFGHIJ operons in microcystin-producing Microcystis strains were revealed [36Noguchi T, Shinohara A, Nishizawa A, et al. Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan J Gen Appl Microbiol 2009; 55: 111-23.
[http://dx.doi.org/10.2323/jgam.55.111]
-38Tooming-Klunderud A, Mikalsen B, Kristensen T, Jakobsen KS. The mosaic structure of the mcyABC operon in Microcystis Microbiology 2008; 154: 1886-99.
[http://dx.doi.org/10.1099/mic.0.2007/015875-0]
], no correlation between possession of the mcy gene and the phylogeny of 16S-23S ITS in domestic Microcystis isolates was observed [36Noguchi T, Shinohara A, Nishizawa A, et al. Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan J Gen Appl Microbiol 2009; 55: 111-23.
[http://dx.doi.org/10.2323/jgam.55.111]
, 37Nishizawa T, Nishizawa A, Asayama M, Harada K-I, Shirai M. Diversity within the microcystin biosynthetic gene clusters among the genus Microcystis Microbes Environ 2007; 22: 380-90.
[http://dx.doi.org/10.1264/jsme2.22.380]
]. It was also found that the mcy gene cluster invariably is arranged between dnaN and uma1 in mcy-possessing Microcystis, whereas dnaNuma1 and hypXuma1 are aligned in non-mcy-possessing Microcystis [37Nishizawa T, Nishizawa A, Asayama M, Harada K-I, Shirai M. Diversity within the microcystin biosynthetic gene clusters among the genus Microcystis Microbes Environ 2007; 22: 380-90.
[http://dx.doi.org/10.1264/jsme2.22.380]
]. Therefore, PCR amplification of mcy was performed using the DNA samples KS2P and KS4P in order to verify the presence of mcy-possessing Microcystis. The region amplified was selected so as to target the region between the 3’-end of the mcyB gene and 5’-end of the mcyC gene (mcyB-C) [6Nishizawa T, Asayama M, Fujii K, Harada K-I, Shirai M. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp J Biochem 1999; 126: 520-9.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481]
] and the NRPS module of mcyG, which is involved in the biosynthesis of a starter unit, Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid), in microcystin [7Nishizawa T, Ueda A, Asayama M, et al. Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin J Biochem 2000; 127: 779-89.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022670]
]. In contrast, to detect microcystin non-producing Microcystis, amplification of the non-coding region between dnaN and uma1 and between hypX and uma1 was conducted as described by Noguchi et al. [36Noguchi T, Shinohara A, Nishizawa A, et al. Genetic analysis of the microcystin biosynthesis gene cluster in Microcystis strains from four bodies of eutrophic water in Japan J Gen Appl Microbiol 2009; 55: 111-23.
[http://dx.doi.org/10.2323/jgam.55.111]
]. According to the PCR analyses of these regions in each gene, no PCR amplicon of mcyG, mcyB-C region, and its non-cording region was observed in KS2P and KS4P (Fig. 2), indicating that an unknown non-mcy-possessing Microcystis was present in the gastric content of the egrets which died.

Sudden appearance of toxic cyanobacteria breaks out in waterbodies. Dynamics of toxic Microcystis population in lake observed temporal changes on the seasonal variability [39Yoshida M, Yoshida T, Takashima Y, Hosoda N, Hiroishi S. Dynamics of microcystin-producing and non-microcystin-producing Microcystis populations is correlated with nitrate concentration in a Japanese lake FEMS Microbiol Lett 2007; 266: 49-53.
[http://dx.doi.org/10.1111/j.1574-6968.2006.00496.x]
]. There has been no report that the wild birds died in the pond in Nishinomiya, Hyogo prefecture, since 1995. Similarly, no deaths of wild birds have been reported since 2008 even though cyanobacterial waterblooms consisting mainly of Microcystis were observed in the reservoir. Many deaths of egrets and herons which causes with the incidence of steatitis have been reported in USA to date. It was suggested that oxidized polyunsaturated fatty acids and toxic cyanobacteria are a possible cause of steatitis [14Rattner AB, McGowan CP. Potential hazards of environmental contaminants to avifauna residing in the Chesapeake Bay estuary Waterbirds 2007; 30: 63-81.
[http://dx.doi.org/10.1675/1524-4695(2007)030[0063:PHOECT]2.0.CO;2]
, 40Wong E, Mikaelian I, Desnoyers M, Fitzgerald G. Pansteatitis in a free-ranging Red-tailed Hawk (Buteo jamaicensis) J Zoo Wildl Med 1999; 30: 584-6.]. Rattner and McGowan [14Rattner AB, McGowan CP. Potential hazards of environmental contaminants to avifauna residing in the Chesapeake Bay estuary Waterbirds 2007; 30: 63-81.
[http://dx.doi.org/10.1675/1524-4695(2007)030[0063:PHOECT]2.0.CO;2]
] reported that microcystins were detected in water samples in Chesapeake Bay and in tissue samples of dead herons, and speculated that microcystins were responsible for the death from steatitis of wild birds. However, microcystins have not yet been proved to cause steatitis. Neagari et al. [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
] reported that no common food items affected with steatitis find in dead egrets and herons in the reservoir because their stomachs were mostly empty, and observed that focal necroses found in liver sections of egret with steatitis. Acute toxic deaths occurred within 3 hours when a fatal dose of microcystin were intraperitoneally (i.p.) injected into mouse [41Nakano M, Nakano Y, Saito-Taki T, et al. Toxicity of Microcystis aeruginosa K-139 strain Microbiol Immunol 1989; 33: 787-92.
[http://dx.doi.org/10.1111/j.1348-0421.1989.tb00964.x]
, 42Ohtake A, Shirai M, Aida T, et al. Toxicity of Microcystis species isolated from natural blooms and purification of the toxin Appl Environ Microbiol 1989; 55: 3202-7.]. The liver of the mouse which died hypertrophied by bleeding, and a necrosis of the liver has occurred with disruption of the architecture of the hepatic cord. Yoshida et al. [43Yoshida T, Makita Y, Nagata S, et al. Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice Nat Toxins 1997; 5: 91-5.
[http://dx.doi.org/10.1002/nt.1]
] reported that median lethal dose (LD50) of the orally given microcystin estimated is 167 times higher than the i.p. LD50 value. Orally administrated microcystin caused primarily hepatocellular injuries with necrosis [43Yoshida T, Makita Y, Nagata S, et al. Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice Nat Toxins 1997; 5: 91-5.
[http://dx.doi.org/10.1002/nt.1]
]. Therefore, it is seemed that microcystin is a possible cause of necroses in the egret with steatitis. Pathologic finding of liver sections in the egret died, does not indicate the acute toxicity by microcystin. To our knowledge, no chronic toxicity by microcystin has been reported. Moreover, microcystins inhibit the protein phosphatase 1 and 2A and act as tumor promoters in human liver [4Nishiwaki-Matsushima R, Ohta T, Nishiwaki S, et al. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR J Cancer Res Clin Oncol 1992; 118: 420-4.
[http://dx.doi.org/10.1007/BF01629424]
, 44Carmichael WW. Toxic Microcystis and the environment. Boca Raton, Florida: CRC Press 1996; pp. 1-11.]. Extracted microcystin-producing Microcystis cells are capable of eliciting a production of interleukin-1 (IL-1) [41Nakano M, Nakano Y, Saito-Taki T, et al. Toxicity of Microcystis aeruginosa K-139 strain Microbiol Immunol 1989; 33: 787-92.
[http://dx.doi.org/10.1111/j.1348-0421.1989.tb00964.x]
] and tumor necrosis factor alpha (TNF-a) [42Ohtake A, Shirai M, Aida T, et al. Toxicity of Microcystis species isolated from natural blooms and purification of the toxin Appl Environ Microbiol 1989; 55: 3202-7.]. Additionally, a delayed hypersensitivity reaction occurs by extracted non-toxic Microcystis cells [45Shirai M, Takamura Y, Sakuma H, Kojima M, Nakano M. Toxicity and delayed type hypersensitivity caused by Microcystis blooms from Lake Kasumigaura Microbiol Immunol 1986; 30: 731-5.
[http://dx.doi.org/10.1111/j.1348-0421.1986.tb02999.x]
]. Microcystis produce small peptides containing novel non-protein amino acids are synthesized by NRPS: the NRPS gene clusters are responsible for the biosynthesis of protease inhibitor micropeptins [46Nishizawa T, Ueda A, Nakano T, et al. Characterization of the locus of genes encoding enzymes producing heptadepsipeptide micropeptin in the unicellular cyanobacterium Microcystis J Biochem 2011; 149: 475-85.
[http://dx.doi.org/10.1093/jb/mvq150]
] and aeruginosin [47Ishida K, Welker M, Christiansen G, et al. Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria Appl Environ Microbiol 2009; 75: 2017-26.
[http://dx.doi.org/10.1128/AEM.02258-08]
] in the genus Microcystis were confirmed using the gene knockout. NRPS and PKS genes of unknown function have been identified in Microcystis as well [19Nishizawa T, Nishizawa A, Shirai M. The potential of natural secondary metabolites produced by cyanobacteria: Genetic approaches to diversify peptide and polyketide biosynthesis. Kerala: Research Signpost 2008; pp. 109-21., 48Nishizawa A, Arshad AB, Nishizawa T, et al. Cloning and characterization of a new hetero-gene cluster of nonribosomal peptide synthetase and polyketide synthase from the cyanobacterium Microcystis aeruginosa K-139 J Gen Appl Microbiol 2007; 53: 17-27.
[http://dx.doi.org/10.2323/jgam.53.17]
]. Furthermore, cyanobacteria, including microcystin-producing strains, produce a large number of peptide compounds with varying bioactivities [49Harada K-I. Production of secondary metabolites by freshwater cyanobacteria Chem Pharm Bull (Tokyo) 2004; 52: 889-99.
[http://dx.doi.org/10.1248/cpb.52.889]
, 50Welker M, von Döhren H. Cyanobacterial peptides–nature's own combinatorial biosynthesis FEMS Microbiol Rev 2006; 30: 530-63.
[http://dx.doi.org/10.1111/j.1574-6976.2006.00022.x]
]. The filamentous cyanobacterium Cylindrospermopsis produces hepatotoxin (i.e. cylindrospermopsin) [51Harada K-I, Ohtani I, Iwamoto K, et al. Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its screening method Toxicon 1994; 32: 73-84.
[http://dx.doi.org/10.1016/0041-0101(94)90023-X]
, 52Ohtani I, Moore RE, Runnegar MT. Cylindrospermopsin, a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii J Am Chem Soc 1992; 114: 7941-2.
[http://dx.doi.org/10.1021/ja00046a067]
], which also inhibits pyrimidine synthesis [53Reisner M, Carmeli S, Werman M, Sukenik A. The cyanobacterial toxin cylindrospermopsin inhibits pyrimidine nucleotide synthesis and alters cholesterol distribution in mice Toxicol Sci 2004; 82: 620-7.
[http://dx.doi.org/10.1093/toxsci/kfh267]
]. Some unknown product of cyanobacteria other than microcystins may have been a cause of the symptoms of steatitis. In addition, we expected that some pesticide residues are concerned with steatitis. As a result of investigating 329 kinds of pesticide residues in an egret which died in the reservoir on Oct. 2008, no pesticide residue was detected to date (Y. Neagari unpublished data).

CONCLUSION

Our results revealed that the dead egrets took in water containing cyanobacterium Microcystis and showed that microcystin was not associated directly with the death of at least two wild egrets. To our knowledge, this study is the first to show the profiling and traceability of a cyanobacterial community in the gastric contents of wild birds with symptoms of steatitis using a molecular analysis. Although necrosis which may be caused by hepatotoxin was observed in the dead egret’s liver [15Neagari Y, Arii S, Udagawa M, et al. Steatitis in egrets and herons from Japan J Wildl Dis 2011; 47: 49-55.
[http://dx.doi.org/10.7589/0090-3558-47.1.49]
], the association between exposures to other bioactive compounds produced by cyanobacteria and the symptoms of steatitis needs to be elucidated in order to verify the cause of death.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

We are grateful to Masayasu Nakano (Emeritus professor of Jichi Medical University) for the valuable comments and helpful suggestions. We also thank Yuko Watanabe, Hiroki Futawatari, and Akira Takanezawa of the laboratory of M.S. for excellent technical assistance. This work was supported in part by a grant from the Mitsui & Co., Ltd. Environment Fund, Japan (to Yasuko Neagari, Koichi Murata, and Ken-Ichi Harada).

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Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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