paralytic shellfish toxin-producing marine dinoflagellatesfile.iocwestpac.org/habs/19-22 dec...

www.harmfulalgae.info

Paralyt ic shel l f i sh tox in-producing MARINE DINOFLAGELLATES


OUTLINES• The organisms (distribution, taxonomy)• The biology and ecology (life‐histories, growth physiology, bloom dynamics, inter‐relationships to other organisms – bacteria, zooplankton, parasites)


The causative organisms of paralytic shellfish poisoning

What we know about


Zonneveld et al. (2013) Review of Palaeobotany and Palynology

Usup et al. (2012) Harmful Algae

Pyrodinium bahamense Global distribution…


Sources:AlexandriumMatsuoka et al., 1997Yoshida et al., 2000Usup et al., 2002Bajarias et al., 2003Nguyen et al., 2004Lim et al., 2004Lim et al., 2005PyrodiniumTing and Wong, 1989Usup et al., 1989Furio and Gonzales, 2002GymnodiniumFukuyo et al., 1993Holmes et al., 2002Mohammand-Nor et al.,2002

Regional distribution…

Fukuyo et al. (2011)


The Taxonomy… Pyrodinium bahamense

Gymnodinium catenatum


The Taxonomy…Alexandrium tamiyavanichii

• A. tamiyavanichii– sym A. cohorticula?

• A. minutum

• A. catenella (A. pacificum?)– Reexamination of specimens from

type locality – Thorough molecular evidence


The molecular systematics

• Morphological plasticity (genetics, environment adaptation)

• Molecular evidence – Which gene marker is suitable?– How many gene marker is sufficient?


• Techniques: – Specific probes with whole‐cell FISH, – qPCR, – flow cytometry

Kon et al. (2015)

Species detection tools


The Life histories in relation to the bloom dynamics

What we know about


Life‐history stages

Technical Guide to Modern Dinoflagellate Cyst Study (Matsuoka & Fukuyo 2000)

Redrawn from Matsuoka & Fukuyo 2000


• Studies on Pyrodinium cyst beds in the Philippines (Azanza1997; 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.


Alexan

drium

(cells L‐1 )

3 × 107

4 × 106

1 × 106

1 × 105

1 × 107

Law et al. (in prep)

Survey results from the 2015 bloom event


Nutrient influenceTetraselmis Diatom

bloom was associated with somewhat lower N/P


Characterizing the phenomenology of bloom event

• A flow cytometry approach• To track the different life history stages and processes affecting bloom initiation and termination.


Diagram adapted from Brosnahan et al. (2013)

Flow cytometry: measuring the DNA content of bloom populations‐ To discern the life cycle stages of the algae at cellular level

Qua

ntiz

ed p

atte

rn


1C

2C4C

1C

2C

4C

1C

2C

4C

Nuclei‐Particles having SG‐associatedfluorescence


1C2C

4C

Relative frequency distribution plots of the field samples recorded in the first bloom


What cause gamete expression and planozygote formation?

Salinity dropped

Temperature decreased


Samples containing 4C cells were likely to reach high abundances at elevated temperatures and low PO4 concentrations.

Canonical Correspondence Analysis


S1

S2

S4

S6

1C

4C

On Aug 31 2015 (when bloom started)


S1

S2

S4

S6

on Sept 27 2015

The bloom was sustained byThe flux of newly-formed vegetative cells.


Lau et al. (in prep)


Liow et al. (in prep)

Sexual processes and Life-history stages

Laboratory setup


Gamete expression and planozygote formation


GROWTH PHYSIOLOGY


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.)


0 20 40 60 80 100 120 1400.0

0.1

0.2

0.3

0.4

0.5 ar2 = 1.00

r2 = 0.99

0 20 40 60 80 100 120 1400.0

0.1

0.2

0.3

0.4

0.5

20o C25o C

b r2 = 0.98

r2 = 0.90

Gro

wth

rate

, (d

ay-1

)

Irradiance (mol photonsm-2s-1)

A. tamiyavanichii A. minutum

Light-adapted

Shade-adapted

Radiating type Peripheral type

Growth physiologyAlexandrium spp.


“Omic” Approaches Achieving the capabilities by Genetic and Metabolic Engineering

• Characterize the physiological responses via “omic” technologies (Transcriptomic, Genomic, metabolomic, etc.)


By investigating the transcriptional responses of the core sxt genes in the saxitoxin biosynthesis… Clonal batch cultures

RNA extraction, cDNA with Superscripts III

Amplification and sequencing

In silico primer design

Relative qPCR: 2‐∆∆CT method

Toxin analysis

• Highest toxin cell quota, Qtwas observed in low P cultures.

• Highest Qt in ammonia‐growth cultures compared to nitrate.

Cell density increased in highest P:NIP>NAP>NLP (Nitrate), AP1> AP2 (Ammonia)• No significant differences were

observed in the mean exponential‐growth rates among treatments.

• Cells adjusted their cellular activities and mechanisms, such that their growth was maintained under various nutrient levels and ratios.


Regulation of the sxtgenes in a tropical Alexandrium minutumstrain is responsive to distinct physiological stresses. This cellular response might be advantageous in unfavorable environmental conditions, thus leading to successful bloom formation.


AmGSIII plays an important role to remove the excess ammonium stress and simultaneously enhance STX production.

Transcriptional and Physiological Responses to Inorganic Nutrition in Alexandriumminutum: Implications for Nutrient Uptakes and Assimilation

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;AmAmt1 is supressed at excess NH4

+ , but AmNrt2 and AmNas were highly expressed.

AmCPSII is related to arginine metabolism and increase the toxin production of the cells.

Survival strategy for A. minutum under unfavorable environment (P‐stressed) is likely inducing the expression of AmNrt2, AmAmt1, AmNas, AmPiPT1 and AmGSIII.


Chal lenges

Gaps


THANK YOU

AcknowledgementsTravel awards by JFIT, WESTPACGrants by MoHE, MOSTI, UMJSPS COMSEAOversea Research AssociatesGraduate students

paralytic shellfish toxin-producing marine dinoflagellatesfile.iocwestpac.org/habs/19-22 dec...

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