www.harmfulalgae.info Pa ra lytic she llfish to xin-p ro d uc ing MARI NE DI NOF L AGE L L AT E S
www.harmfulalgae.info OUTLINES • The organisms (distribution, taxonomy) • The biology and ecology (life‐histories, growth physiology, bloom dynamics, inter‐ relationships to other organisms – bacteria, zooplankton, parasites)
www.harmfulalgae.info What we kno w abo ut The causative organisms of paralytic shellfish poisoning
www.harmfulalgae.info Glo bal distributio n… Pyrodinium bahamense Zonneveld et al. (2013) Review of Palaeobotany and Palynology Usup et al. (2012) Harmful Algae
www.harmfulalgae.info Re g io nal distributio n… Fukuyo et al. (2011) Sources: Alexandrium Matsuoka et al., 1997 Yoshida et al., 2000 Usup et al., 2002 Bajarias et al., 2003 Nguyen et al., 2004 Lim et al., 2004 Lim et al., 2005 Pyrodinium Ting and Wong, 1989 Usup et al., 1989 Furio and Gonzales, 2002 Gymnodinium Fukuyo et al., 1993 Holmes et al., 2002 Mohammand-Nor et al., 2002
www.harmfulalgae.info T he T axo no my… Pyrodinium bahamense Gymnodinium catenatum
www.harmfulalgae.info T he T axo no my… Alexandrium tamiyavanichii • A. tamiyavanichii – sym A. cohorticula ? • A. minutum • A. catenella (A. pacificum?) – Reexamination of specimens from type locality – Thorough molecular evidence
www.harmfulalgae.info T he mo le c ular syste matic s • Morphological plasticity (genetics, environment adaptation) • Molecular evidence – Which gene marker is suitable? – How many gene marker is sufficient?
www.harmfulalgae.info Spe c ie s de te c tio n to o ls • Techniques: – Specific probes with whole‐cell FISH, – qPCR, – flow cytometry Kon et al. (2015)
www.harmfulalgae.info What we kno w abo ut The Life histories in relation to the bloom dynamics
www.harmfulalgae.info Life‐history stages Technical Guide to Modern Dinoflagellate Cyst Study (Matsuoka & Fukuyo 2000) Redrawn from Matsuoka & Fukuyo 2000
www.harmfulalgae.info • Studies on Pyrodinium cyst beds in the Philippines (Azanza 1997; Azanza et al. 1999, 2004), Ambon Bay, Indonesia (Mizushima et al. 2007), Sabah, Malaysia (Furio et al. 2012) • Alexandrium cyst studies in Japan (e.g. Natsuike et al. 2013; Kamiyama et al. 2014) • Limited data on the relationship of life‐history stages and the bloom dynamics in the SEA.
www.harmfulalgae.info
www.harmfulalgae.info Surve y re sults fro m the 2015 blo o m e ve nt Alexandrium (cells L ‐1 ) 3 × 10 7 1 × 10 7 4 × 10 6 1 × 10 6 1 × 10 5 Law et al. (in prep)
www.harmfulalgae.info Nutrie nt influe nc e Diato m T e trase lmis blo o m was asso c iate d with so me what lo we r N/ P
www.harmfulalgae.info Charac te rizing the phe no me no lo g y o f blo o m e ve nt • A flow cytometry approach • To track the different life history stages and processes affecting bloom initiation and termination.
www.harmfulalgae.info Flow cytometry: measuring the DNA content of bloom populations ‐ To discern the life cycle stages of the algae at cellular level Quantized pattern Diagram adapted from Brosnahan et al. (2013)
www.harmfulalgae.info Nuclei‐ Particles having 4C 2C SG‐associated fluorescence 1C 2C 4C 4C 2C 1C 1C
www.harmfulalgae.info 2C 1C 4C Relative frequency distribution plots of the field samples recorded in the first bloom
www.harmfulalgae.info What c ause game te e xpre ssio n and plano zygo te fo rmatio n? Salinity dro ppe d T e mpe rature de c re ase d
www.harmfulalgae.info Samples containing 4C cells were likely to reach high abundances at elevated temperatures and low PO 4 concentrations. Canonical Correspondence Analysis
www.harmfulalgae.info 1C S1 S2 S4 4C S6 On Aug 31 2015 (whe n blo o m starte d)
www.harmfulalgae.info S1 S2 S4 T he blo o m was sustaine d by S6 T he flux o f ne wly-fo rme d ve g e tative c e lls. o n Se pt 27 2015
www.harmfulalgae.info Lau et al. (in prep)
www.harmfulalgae.info Se xual pro c e sse s and L ife -histo ry stag e s L abo rato ry se tup Liow et al. (in prep)
www.harmfulalgae.info Game te e xpre ssio n and plano zygo te fo rmatio n
www.harmfulalgae.info GROWTH PHYSIOLOGY
www.harmfulalgae.info Growth physiology • Pyrodinium bahamense (Usup et al. 1994; Gedaria et al. 2007; reviewed in Usup et al. 2012) • Alexandrium spp. (e.g. Ogata et al. 1999, 2001; Wang et al. 2008 etc.)
www.harmfulalgae.info Growth physiology Alexandrium spp. Radiating type Peripheral type A. minutum A. tamiyavanichii 0.5 a Growth rate, (day -1 ) b 0.5 r 2 = 0.98 Light-adapted 0.4 0.4 r 2 = 1.00 r 2 = 0.90 0.3 0.3 r 2 = 0.99 Shade-adapted 0.2 0.2 20 o C 0.1 0.1 25 o C 0.0 0.0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Irradiance ( mol photons m -2 s -1 )
www.harmfulalgae.info “Omic” Approaches Achieving the capabilities by Genetic and Metabolic Engineering • Characterize the physiological responses via “omic” technologies (Transcriptomic, Genomic, metabolomic, etc.)
www.harmfulalgae.info By investigating the transcriptional responses of the core sxt genes in the saxitoxin biosynthesis… Clonal batch cultures Toxin analysis Cell density increased in highest P: NIP>NAP>NLP (Nitrate), AP1> AP2 (Ammonia) • No significant differences were RNA extraction, observed in the mean exponential ‐ cDNA with Superscripts III growth rates among treatments. • Cells adjusted their cellular activities and mechanisms, such In silico primer design that their growth was maintained under various nutrient levels and ratios . Amplification and sequencing • Highest toxin cell quota, Q t was observed in low P cultures. • Highest Q t in ammonia ‐ growth cultures compared Relative qPCR: 2 ‐∆∆ C T method to nitrate.
www.harmfulalgae.info Regulation of the sxt genes in a tropical Alexandrium minutum strain is responsive to distinct physiological stresses. This cellular response might be advantageous in unfavorable environmental conditions, thus leading to successful bloom formation.
www.harmfulalgae.info Transcriptional and Physiological Responses to Inorganic Nutrition in Alexandrium minutum : Implications for Nutrient Uptakes and Assimilation Survival strategy for A. minutum under unfavorable environment (P‐stressed) is likely inducing the expression of AmNrt2 , AmAmt1 , AmCPSII is related to arginine AmNas , AmPiPT1 and metabolism and increase the toxin AmGSIII. production of the cells. AmGSIII plays an important role to remove the excess ammonium stress and simultaneously enhance STX production. AmNrt2, AmAmt1 and AmPiPT1 are high affinity transporters; AmGSIII is a mitochondria enzyme; AmNas is a cytosol enzymes; and AmCPSII is a pyrimidines synthesis related enzyme. Uptake of nitrate is favourable than ammonium in A . minutum; + , but AmNrt2 and AmNas were highly expressed. AmAmt1 is supressed at excess NH 4
C ha lle ng e s www.harmfulalgae.info G a p s
www.harmfulalgae.info Acknowledgements Travel awards by JFIT, WESTPAC Grants by MoHE, MOSTI, UM JSPS COMSEA Oversea Research Associates Graduate students THANK YOU
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