Toxicity and Protein Expression of Alexandrium Species Collected in the Philippine Waters

Bryan John J. Subong1, Garry A. Benico1, Arielle Kae L. Sulit1a,
Christopher O. Mendoza1b, Lourdes J. Cruz1, Rhodora V. Azanza1, and Elsie C. Jimenez2*

1The Marine Science Institute, College of Science,
University of the Philippines Diliman, Quezon City 1101, Philippines
2Department of Physical Sciences, College of Science,
University of the Philippines Baguio, Baguio City 2600, Philippines
Present addresses:
aSt. Luke’s Medical Center, E. Rodriguez Sr. Ave., Quezon City 1112, Philippines
bPhilippine Nuclear Research Institute, Commonwealth Ave., Diliman, Quezon City 1101, Philippines

*Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.


Isolates of Alexandrium species collected in the Philippine waters were examined during the exponential growth phase to compare their toxicities and protein expression profiles, and also to correlate protein expression with toxin production. Molecular methods showed that Alexandrium cf. pacificum was genetically divergent from Alexandrium tamarense complex Group IV/ Alexandrium pacificum. Toxin analyses using pre-oxidation method prior to HPLC purification were carried out to determine saxitoxin (STX), neosaxitoxin (neoSTX), and gonyautoxins 1-4 (GTX) levels. This study showed that cultured Alexandrium affine collected in Honda Bay, Palawan produced different STX analogs at various times of culture, which differed from other results showing that this species was non-toxic. The cultured Alexandrium cf. pacificum collected in Anda Channel (ATANDA) was two-fold more toxic than the cultured Alexandrium cf. pacificum collected in Bolinao Channel (ATBOL). Comparative protein expression analyses using 2-D gel electrophoresis were performed for the two Alexandrium cf. pacificum strains (ATANDA and ATBOL) during the exponential growth phase. A unique 2-DE protein spot in ATANDA showed sequence homology with bifunctional ornithine acetyltransferase/ N-acetylglutamate synthase (ArgJ) that has a role in the biosynthesis of arginine, a precursor in STX biosynthesis. The greater detectable expression of such enzyme in the ATANDA strain was correlated with the greater toxicity, suggesting the enzyme’s major participation in toxin biosynthesis.

Research efforts have been geared towards the understanding of the major causative agents of paralytic shellfish poisoning (PSP) in the Philippines due to the numerous cases of PSP leading to human fatalities. It has been reported that majority of PSP cases in Southeast Asia occurred in the Philippines due mainly to blooms of dinoflagellates such as Pyrodinium bahamense and Alexandrium sp. (Yñiguez et al. 2012, Azanza & Benico 2013). . . . . read more

AGUILERA-BELMONTE A, INOSTROZA I, FRANCO JM, RIOBÓ P, GÓMEZ PI. 2011. The growth, toxicity and genetic characterization of seven strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated during the 2009 summer outbreak in southern Chile. Harmful Algae 12:105-112.
ALTSCHUL S, GISH W, MILLER W, MYERS E, LIPMAN D. 1990. Basic local alignment search tool (BLAST). J Mol Biol 215(3):403-410.
ANDERSON DM, ALPERMANN TJ, CEMBELLA  AD, COLLOS Y, MASSERET E, MONTRESSOR M. 2012. The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae 14:10-35.
AZANZA RV, BENICO GA. 2013. Toxic Alexandrium blooms in fish farming sites in Bolinao, Pangasinan. J Environ Sci Manage 1-2013:44-49.
AZANZA-CORRALES R, HALL S. 1993. Isolation and culture of Pyrodinium bahamense var. compressum from the Philippines.  In: Toxic Phytoplankton Blooms in the Sea. Smayda TJ, Shimizu Y ed. Amsterdam: Elsevier Science Publishers BV. p. 725-730.
BALECH E. 1995. The genus Alexandrium Halim (Dinoflagellata). Sherkin Island Marine Station, Cork, Ireland. 151p.
BALLOT A, BERNARD C, FASTNER J. 2016. Saxitoxin and Analogues. In: Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis. Meriluoto J, Spoof L, Codd GA ed. United States of America: John Wiley & Sons. p. 148-153.
BAND-SCHMIDT CJ, LECHUGA-DEVEZE CH, KULIS DM, ANDERSON DM. 2003. Culture studies of Alexandrium affine (Dinophyceae), a non-toxic cyst forming dinoflagellate from Bahía Concepción, Gulf of California. Botanica Marina 46(1):44-54.
CHAN LL, HODGKISS IJ, LU S, LO SC. 2004. Use of two-dimensional gel electrophoresis proteome reference maps of dinoflagellates for species recognition of causative agents of harmful algal blooms. Proteomics 4(1):180-192.
CHAN LL, HODGKISS IJ, LAM PK, WAN JM, CHOU HN, LUM JH, LO MG, MAK AS, SIT WH, LO SC. 2005. Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms. Proteomics 5(6):1580-1593.
CHAN LL, SIT WH, LAM PK, HSIEH DP, HODGKISS IJ, WAN JM, HO AY, CHOI NM, WANG DZ, DUDGEON D. 2006. Identification and characterization of a “biomarker of toxicity” from the proteome of the paralytic shellfish toxin-producing dinoflagellate Alexandrium tamarense (Dinophyceae). Proteomics 6(2):654-666.
CRABEEL M, ABADJIEVA A, HILVEN P, DESIMPELAERE J, SOETENS O. 1997. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity. Eur J Biochem 250(2):232-241.
DELL’AVERSANO C, WALTER JA, BURTON IW, STIRLING DJ, FATTORUSSO E, QUILLIAM MA. 2008. Isolation and structure elucidation of new and unusual saxitoxin analogues from mussels. J Nat Prod 71:1518-1523.
FRITZ L, TRIEMER RE. 1985. A rapid technique utilizing Calcofluor White M2R for the visualization of dinoflagellate thecal plates. J Phycol 21(4):662-664.
FUKUYO Y. 2001. Research on the Ecology and Taxonomy of PSP Toxin-Producing Alexandrium. Asian Biological Resource and Environment Research Center, Tokyo University. 17:2001.
GROMOVA I, CELIS JE. 2006. Protein detection in gels by silver staining: a procedure compatible with mass-spectrometry. In: Cell Biology: A Laboratory Handbook, 4th ed. Celis JE, Carter N, Hunter T, Simon K, Small JV, Shotton D ed. United States of America: Elsevier Academic Press. p. 219-223.
GUILLARD RL, RYTHER JH. 1962. Studies of marine planktonic diatoms I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can J Microbiol 8(2):229-239.
HAMASAKI K, HORIE M, TOKIMITSU S, TODA T, TAGUCHI S. 2001. Variability in toxicity of the dinoflagellate Alexandrium tamarense isolated from Hiroshima Bay, western Japan, as a reflection of changing environmental conditions. J Plankton Res 23(3):271-278.
HAN M, LEE H, ANDERSON DM, KIM B. 2016. Paralytic shellfish toxin production by the dinoflagellate Alexandrium pacificum (Chinhae Bay, Korea) in axenic, nutrient-limited chemostat cultures and nutrient-enriched batch cultures. Marine Poll Bull 104(1):34-43.
HII KS, LIM PT, KON NF, TAKATA Y, USUP G, LEAW CP. 2016. Physiological and transcriptional responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for the saxitoxin genes and toxin production. Harmful Algae 56:9-21.
JIANG XW, WANG J, GAO Y, CHAN LL, LAM PKS, GU JD. 2015. Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation. Ecotoxicology 24(7-8):1744-1753.
JOHN U, LITAKER W, MONTRESOR M, MURRAY S, BROSNAHAN ML, ANDERSON DM. 2014. Proposal to reject the name Gonyaulax catenella (Alexandrium catenella) (Dinophyceae). Taxon 63(4):932-933.
KELLMANN R, MIHALI TK, JEON YJ, PICKFORD R, POMATI F, NEILAN BA. 2008. Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria. Applied Env Microbiol 74(13):4044-4053.
KRUGER NJ. 2009. The Bradford method for protein quantitation.  In: The Protein Protocols Handbook, 3rd ed Walker J ed. United States of America: Humana Press. p.17-24.
LAWRENCE JF, MÉNARD C. 1991. Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechromatographic oxidation. J Assoc Anal Chem 74(6):1006-1012.
LAWRENCE JF, NIEDZWIADEK B. 2001. Quantitative determination of paralytic shellfish poisoning toxins in shellfish by using prechromatographic oxidation and liquid chromatography with fluorescence detection. J Assoc Anal Chem Intl 84(4): 1099-1108.
LEE FW, LO SC. 2008. The use of Trizol reagent (phenol/guanidine isothiocyanate) for producing high quality two-dimensional gel electrophoretograms (2-DE) of dinoflagellates. J Microbiol Methods 73(1):26-32.
LILLY EL, HALANYCH KM, ANDERSON DM. 2007. Species boundaries and global biogeography of the Alexandrium tamarense complex (Dinophyceae). J Phycol 43(6):1329-1338.
LOGARES R, SHALCHIAN-TABRIZI K, BOLTOVSKOY A, RENGEFORS K. 2007. Extensive dinoflagellate phylogenies indicate infrequent marine-freshwater transitions. Mol Phylogenet Evol 45(3):887-903.
MARC F, WEIGEL P, LEGRAIN C, ALMERAS Y, SANTROT M, GLANSDORFF N, SAKANYAN V. 2000. Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms. Eur J Biochem 267(16):5217-5226.
NANDAKUMAR MP, SHEN J, RAMAN B, MARTEN MR. 2002. Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via NaOH for two-dimensional electrophoresis. J Proteome Res 2(1):89-93.
NGUYEN-NGOC L. 2004. An autecological study of the potentially toxic dinoflagellate Alexandrium affine isolated from Vietnamese waters. Harmful Algae 3(2):117-129.
ONDA DFL, BENICO G, SULIT AF, GAITE PL, AZANZA RV, LLUISMA AO.  2013. Morphological and molecular characterization of some HAB forming dinoflagellates from Philippine waters. Phil Sci Lett. 6(1):97-106.
OSHIMA Y. 1995. Postcolumn derivatization liquid chromatographic method for paralytic shellfish toxins. J Assoc Anal Chem Intl 78(2):528-532.
PARKINSON JE, BAUMGARTEN S, MICHELL CT, BAUMS IB, LAJEUNESSE TC, VOOLSTRA CR. 2016. Gene expression variation resolves species and individual strains among coral-associated dinoflagellates within the genus Symbiodinium. Genome Biol Evol 8(3):665-680.
PEAKS (Version 7.0.2) [Computer software]. (2014). Waterloo, ON, Canada: Bioinformatics Solutions. Available from
PERSICH GR, KULIS DM, LILLY EL, ANDERSON DM, GARCIA VMT. 2006. Probable origin and toxin profile of Alexandrium tamarense (Lebour) Balech from southern Brazil. Harmful Algae 5(1):36-44.
SAKANYAN V, CHARLIER D, LEGRAIN C, KOCHIKYAN A, METT I, PIÉRARD A, GLANSDORFF N. 1993. Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase. J Gen Microbiol 139(3):393-402.
SHEVCHENKO A, SUNYAEV S, LOBODA A, SHEVCHENKO A, BORK P, ENS W, STANDING KG. 2001. Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching. Anal Chem 73(9):1917-1926.
SHEVCHENKO A, SUNYAEV S, LISKA A, BORK P, SHEVCHENKO A.  2003. Nanoelectrospray tandem mass spectrometry and sequence similarity searching for identification of proteins from organisms with unknown genomes. Methods Mol Biol 211:221-234.
SHEVCHENKO A, TOMAS H, HAVLI J, OLSEN JV, MANN M. 2006. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856-2860.
SHIMIZU Y, NORTE M, HORI A, GENENAH A, KOBAYASHI M. 1984. Biosynthesis of saxitoxin analogs: the unexpected pathway. J Amer Chem Soc 106(21):6433-6434.
SHIMIZU Y. 1993. Microalgal metabolites. Chem Rev 93(5):1685-1698.
SZKLARCZYK D, FRANCESCHINI A, WYDER S, FORSLUND K, HELLER D, HUERTA-CEPAS J, KUHN M. 2014. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:447-452.
TAMURA K, PETERSON D, PETERSON N, STECHER G, NEI M, KUMAR S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731-2739.
TOMAS CR ed. 1997. Identifying marine phytoplankton. United States of America: California Academic Press. 858p.
WANG DZ, HSIEH DP. 2005. Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea. Harmful Algae 4(2):401-410.
WANG DZ, LIN L, CHAN LL, HONG HS. 2009. Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two dimensional electrophoresis. Harmful Algae 8(5):685-691.
WANG DZ, DONG HP, LI C, XIE ZX, LIN L, HONG HS.  2011. Identification and characterization of cell wall proteins of a toxic dinoflagellate Alexandrium catenella using 2-D DIGE and MALDI-TOF mass spectrometry. Evid Based Complement Altern Med 2011:984080.
WANG DZ, GAO Y, LIN L, HONG HS. 2013. Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella. Marine Drugs 11(1):213-232.
YÑIGUEZ AT, CAYETANO A, VILLANOY CL, ALABIA I, FERNANDEZ I, PALERMO, JD, BENICO GA, SIRINGAN FP, AZANZA RV. 2012. Investigating the roles of intrinsic and extrinsic factors in the blooms of Pyrodinium bahamense var. compressum using an individual-based model. Procedia Environ Sci 13:1462-1476.
ZOU C, YE RM, ZHENG JW, LUO ZH, GU HF, YANG WD, LI HY, LIU JS. 2014. Molecular phylogeny and PSP toxin profile of the Alexandrium tamarense species complex along the coast of China. Marine Poll Bull 89(1):209-219.