Morphological characterization and molecular detection of potential harmful diatom, pseudo-nitzschia from Malaysian water

Teng, Sing Tung (2012) Morphological characterization and molecular detection of potential harmful diatom, pseudo-nitzschia from Malaysian water. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

The diatom, Pseudo-nitzschia is a genus that associated with harmful algal blooms (HABs) worldwide. One third of the species produce the neurotoxin, domoic acid (DA) which is a causative agent of amnesic shellfish poisoning (ASP). Research interest in toxic Pseudo-nitzschia thrived after the outbreak of the first ASP case stroked Canada in 1987. Following the incidence, numerous studies have been undertaken throughout the open oceans. However, occurrences of Pseudo-nitzschia spp. were reported as species complex/complexes due to the taxonomic uncertainty in the genus. This has lead to enormous confusion in species identification) In this study, a thorough field survey focused on Pseudo-nitzschia species were undertaken to investigate the spatial distribution pattern and species composition in Malaysian waters. Samplings covered locations in the Andaman Sea, the Straits of Malacca, the South China Sea, Sulu Sea and Celebes Sea. Plankton samples were collected from 17 locations using a 20 gmmesh plankton net haul. Samples were subjected to acid wash treatment before detailed morphological observations under transmission electron microscope (TEM). Identification of Pseudo-nitzschia species was based on the frustules morphology with morphometric measurements. In total, 22 species were identified; in which 16 were new records in Malaysia, and a new morphotype discovered. Among the toxic species found, including P. brasiliana, P. caciantha, P. calliantha, P. cuspidata, P. delicatissima, P. multistriata, P. pseudodelicatissima, P. pungens, and P. turgidula were previously reported to associate with ASP events worldwide. In order to aid the species identification of this complex species, molecular approaches have been introduced in Pseudo-nitzschia taxonomy. Molecular phylogenetic analyses are applied based on a genetic marker to resolve the Pseudonitzschia species complex/complexes. In this study, whole cell fluorescence in situ hybridization (FISH), a rapid molecular detection tool was applied on both field and cultured samples. Two toxic species, P. pungens were used as model species in FISH optimization and on-field application. Species-specific probes were designed in silico. The accessibilities of probes were tested in silico based on complete 28S rRNA secondary structure information. The probes were successfully optimized on clonal cultures and natural spiked samples. Whole-cell FISH was proven as a potential rapid detection tool for monitoring of toxic Pseudo-nitzschia species in Malaysia. This study has provided further insight and better understanding on Pseudo-nitzschia taxonomy using both the morphological and molecular approaches. The finding of a new morphotype warrants further investigation to obtain genetic evidence. FISH method with species specific probes on targeted Pseudo-nitzschia species are proposed as monitoring tools for the related agency in Malaysia.

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