Ultrastructural and Molecular Aspects of Flagella Assembly in the Marine Dinoflagellate Alexandrium Minutum

Doctoral Thesis / Dissertation , 2013 , 185 Pages

Biology - Micro- and Molecular Biology

Dinoflagellates are microscopic unicellular microalgae found in aquatic environments worldwide. One common feature of dinoflagellates is the presence of two unequal flagella: The longitudinal flagellum is round while the transverse flagellum is ribbon-shaped. Currently little is known about the intracellular ultrastructure of the dinoflagellate flagella apparatus and molecular aspects of the flagella assembly are unknown.

In this study a combination of ultrastructure studies using scanning (SEM) and transmission electron microscopy (TEM), and molecular analysis of transcriptomics data was used to gain understanding on the flagella apparatus of the toxic marine dinoflagellate Alexandrium minutum. Samples for SEM and TEM observations were prepared using standard protocols. For transcriptomics analysis, total RNA was harvested from exponential phase cultures during the light and dark culture periods. The transcriptome was sequenced using HiSeq sequencing, assembled and annotated. The assembled sequences were searched for putative genes involved in flagella synthesis, assembly and functioning based on annotations and reverse Blast analysis.

The putative genes were used in phylogenetic and in silico protein analysis. Transcriptome sequencing produced 399,169 unigenes, of which 412 were related to flagella biosynthesis and function. Structural flagella genes found were α, β and γ dynein; α, β and γ tubulin; radial spoke, basal body, central pair wd, central pair pf20, flagellar protofilament ribbon protein, calcium binding, sodium–type flagellar motor component and nexin. There were also several intraflagellar transport (IFT) genes namely 20, 27, 52, 57, 74, 80, 81, 88, 122 and 172.

The other flagella related genes found were genes for kinesin II and cytoplasmic dynein 2. Ultrastructure observation by TEM showed that the Cells had the typical dinoflagellate organelles. The chloroplast contained two or three apprised thylakoids, and was surrounded by three membranes, the eyespot is located dorsally and composed of one or two layers of globules situated within the chloroplast, In the periphery of the cell, trichocysts, mucocysts, and unidentified multilayered material were visible This study provided for the first time a large volume of gene data that can be used to better understand the molecular basis of flagella biosynthesis and function in dinoflagellates.

Table of Contents

• CHAPTER I INTRODUCTION
• 1.1 RESEARCH BACKGROUND
• 1.2 PROBLEM STATEMENT
• 1.3 OBJECTIVES OF RESEARCH AND SCOPE OF WORKS
• CHAPTER II LITERATURE REVIEW
• 2.1 Dinoflagellates
• 2.1.1 General characteristics of dinoflagellate
• 2.1.2 Genetic characteristics
• 2.1.3 Morphology and taxonomy
• 2.1.4 Life cycles
• 2.1.5 Molecular phylogenies
• 2.1.6 Harmful algal blooms
• 2.1.7 Ecology
• 2.1.8 Alexandrium minutum
• 2.2 Eukaryotic flagella apparatus
• 2.2.1 Structure of eukaryotic flagella
• 2.2.2 flagellar assembly
• 2.3 Scanning and transmission electron microscopy in dinoflagellate
• 2.3.1 Scanning electron microscopy (SEM)
• 2.3.2 Transmission electron microscopy (TEM)
• CHAPTER III Material and methods
• 3.1 Cell culture
• 3.1.1 Preparation sterile seawater
• 3.1.2 Preparation medium culture
• 3.1.3 Culturing
• 3.2 Cell harvesting and total RNA extraction
• 3.2.1 Cell harvesting
• 3.2.2 Total RNA extraction
• 3.3 EST Database Analysis and Managemen
• 3.3.1 Pipeline of Experiments
• 3.3.2 Output Statistics
• 3.3.3 Phylogenetic constructions
• 3.4 Microscopy
• 3.4.1 Light microscopy
• 3.4.2 Scanning electron microscopy
• 3.4.3 Transmission electron microscopy
• CHAPTER IV result and discussion
• 4.1 microscopy
• 4.1.1 Light microscop
• 4.1.2 Scanning electron microscopy
• 4.1.3 Transmission electron microscopy
• 4.2 molecular analyses
• 4.2.1 Overview of transcriptome sequencing results
• 4.3 Flagellar unigens
• 4.3.1 Known structural flagellar genes
• 4.3.2 Intraflagellar transport components
• CHAPTER V CONCLUSION AND FUTURE WORKS
• 5.1 CONCLUSION
• 5.2 SUGGESTIONS FOR FUTURE WORK

Objectives and Key Themes

This thesis aims to investigate the ultrastructural and molecular aspects of flagella assembly in the marine dinoflagellate Alexandrium minutum. The research focused on characterizing the flagellar apparatus of this toxic species through a combination of scanning and transmission electron microscopy and transcriptomics data analysis. The study aimed to identify genes related to flagella biosynthesis and function, understand their role in flagella assembly, and gain insights into the molecular basis of flagella function in dinoflagellates.

• Ultrastructural analysis of Alexandrium minutum using electron microscopy.
• Transcriptome sequencing and de novo analysis of Alexandrium minutum.
• Identification and analysis of structural genes involved in flagella biosynthesis and function.
• Investigation of intraflagellar transport genes and their role in flagella assembly.
• Understanding the molecular basis of flagella function in dinoflagellates.

Chapter Summaries

• Chapter I: Introduction This chapter provides background information on dinoflagellates, their importance in marine ecosystems, and the significance of Alexandrium minutum as a toxic species. It also highlights the limited knowledge about flagella assembly in dinoflagellates and outlines the objectives and scope of the research.
• Chapter II: Literature Review This chapter presents a comprehensive overview of dinoflagellate biology, focusing on their general characteristics, genetic features, morphology, life cycles, molecular phylogenies, harmful algal blooms, ecology, and the specific characteristics of Alexandrium minutum. It also delves into the structure and assembly of eukaryotic flagella, including detailed information on dyneins, microtubules, tubulins, radial spokes, basal bodies, and the intraflagellar transport mechanism. Lastly, it discusses the application of scanning and transmission electron microscopy in dinoflagellate research, emphasizing the importance of these techniques in understanding the ultrastructure of this group of organisms.
• Chapter III: Material and Methods This chapter describes the methods used in the study, including cell culture techniques, RNA extraction protocols, EST database analysis, and microscopy methods. It details the specific protocols for preparing samples for scanning and transmission electron microscopy, as well as the bioinformatics analysis of transcriptomics data.
• Chapter IV: Results and Discussion This chapter presents the results obtained through the study, focusing on both ultrastructural and molecular analyses. The chapter provides a detailed description of the ultrastructure of Alexandrium minutum as observed through light microscopy, scanning electron microscopy, and transmission electron microscopy. It also discusses the results of transcriptome sequencing, unigene analysis, and the identification of genes related to flagella biosynthesis and function. The chapter further explores the intraflagellar transport components identified in A. minutum, discussing their potential roles in flagella assembly.

Keywords

The main keywords and focus topics of this thesis include dinoflagellates, Alexandrium minutum, harmful algal blooms, flagella, flagellar assembly, ultrastructure, transcriptomics, gene expression, intraflagellar transport, dynein, tubulin, radial spokes, basal bodies, and molecular biology.

• College: University Kebangsaan Malaysia
• Author: Nahid Khalili (Author)
• Publication Year: 2013
• Pages: 185
• Catalog Number: V1416320
• ISBN (PDF): 9783346966896
• Language: English
• Tags: ultrastructural, molecular, aspects, flagella, assembly, marine, dinoflagellate, alexandrium, minutum