Development of molecular platform for the detection of harmful algal bloom species, Alexandrium minutum / Winnie Lau Lik Sing
Toxic marine dinoflagellate Alexandrium minutum is widely associated with paralytic shellfish poisoning (PSP) in the Asia Pacific region. This species have been reported for PSP cases at east coast of Peninsular Malaysia. In this study, a powerful and advanced method was demonstrated for the dete...
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| Format: | Thesis |
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2017
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| Online Access: | http://studentsrepo.um.edu.my/8299/ http://studentsrepo.um.edu.my/8299/2/All.pdf http://studentsrepo.um.edu.my/8299/6/winnie.pdf |
| Summary: | Toxic marine dinoflagellate Alexandrium minutum is widely associated with
paralytic shellfish poisoning (PSP) in the Asia Pacific region. This species have been
reported for PSP cases at east coast of Peninsular Malaysia. In this study, a powerful
and advanced method was demonstrated for the detection of A. minutum using SYBR
Green I-based quantitative real-time PCR (qPCR) and investigates the life cycle of A.
minutum in natural bloom using flow cytometry. For qPCR detection, species specific
primers were designed from second internal transcribed spacer (ITS2) region of
ribosomal RNA gene (rDNA) which had been showed to be the excellent molecular
signature for species identification and discrimination. The qPCR assay showed high
specificity and sensitivity in detecting A. minutum from the environmental samples.
This assay allow quantification of target cells by convert total extractable gene copies
into cell number based on the extractable gene copy number per cell estimated in the
studies. Gene-based calibration curve with AE 97.6% was served as an alternative
calibrator in A. minutum qPCR quantification. Based on the calibration curve, the assay
estimated 7,584 ± 1,139 extractable gene copies per cell of A. minutum. However, the
gene copy number per cell determined is only an estimate to convert the total
extractable gene copies in a sample to cell estimates. In addition, SYBR Green I-based
qPCR assay was applied to the A. minutum bloom samples. However, overestimation
and underestimation in some samples were observed due to different DNA content at
different stage of life cycle. Specificity and sensitivity of the assay provide an effective
method for A. minutum monitoring especially in the site with different Alexanderium
spp. coexist. In early September 2015, a remarkably high density of A. minutum
occurred in Sungai Geting, Tumpat, a semi-enclosed lagoon at north-eastern Peninsular
Malaysia, causing severe discoloration and contaminated the benthic clam, Polymesoda similis. Samplings were undertaken over the duration of four months between the bloom
periods from September to December 2015. Plankton samples were collected and used
to investigate the mechanisms of blooms initiation, development and termination by
flow cytometry based detection. A 48-h sampling was conducted in March 2016 when
the bloom recurred to investigate the life cycle stage. Results from flow cytometric
analysis showed that planomyocetes (4C) were observed (88%) in the early bloom,
suggesting that the bloom was initiated by cyst excystment. Increase in planozygotes
(2C) was signified in the middle of the bloom, coincided with an abrupt decrease in
salinity and elevated N:P ratio. Gamate expression and mating process were induced
among the population under phosphorous limitation. The results also confirmed that the
bloom was initiated at the inner part of the lagoon and slowly dispersed to the river
mouth. The blooms were sustained for four months by 72 92% of vegetative cells (1C)
although 36% of planozygotes (2C) were observed in the early of the bloom. Results
from the 48-h diurnal sampling confirmed that cells undergo consistent cell cycle in
natural environment. Increase of precipitation and freshwater intrusion from the Golok
River might cause the bloom to terminate. This event provides a fundamental
understanding of the life cycle of this tropical toxic dinoflagellate. |
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