Molecular characterisation of Jatropha Curcas : towards an understanding of its potential as a non-edible oilseed-based source of biodiesel

Jatropha curcas, a non-edible oilseed, is becoming popular as a source of biodiesel. However, there are some limitations to using J. curcas such as the presence of carcinogenic substances, unpredictable yield, asynchronous flowering and plant height. Understanding the genetic control of these characteristics will help to find solutions. To accumulate genetic information for J. curcas plant breeding programmes, the genetic and phenotypic characterisation of J. curcas seeds obtained from different geographical locations was undertaken (Chapter 2). The results showed a narrow genetic diversity among accessions but apparent phenetic diversity. The screens employed ranged from phenotypic characterizations of seeds to molecular, biochemical, physiological and genotyping assessments. Seed dimensions and weight measurements provided data on seed density and kernel weight, both important parameters for assessing oilseed quality. Biochemical analysis provided data on oil properties relevant to biodiesel e.g. total oil content, oleic/linoleic acid ratio, saponification number, cetane number and gross energy value. Expression analysis of genes (acc, sad, fad, and dgat) essential for the quantity and quality of seed/oil were targeted as molecular screens. Understanding the molecular mechanism of lipid cycling in J. curcas seeds will guide future improvements in oil yield. However, this is still an area of active research because little information exists for this in J. curcas. With regards to oil storage capacity of seeds, no information previously existed on the biology of oil bodies in Jatropha curcas. In this study, oil bodies from J. curcas were studied at the gene transcript and protein levels (Chapter 3). Three Jatropha oleosin genes were isolated. Sequence analysis showed that all three oleosin genes contained the proline knot conserved domain. The upstream regulatory element of one Jatropha oleosin gene was also isolated. In silico analysis of this upstream sequence indicated the presence of a number of seed specific regulatory motifs. Transcript analysis for the three oleosin genes revealed tissue-specific differences. Immunoassay by western blot analysis using antibodies against Arabidopsis oleosin and the J. curcas specific oleosin revealed a diversity of oleosins. Proteomic analysis of oil bodies revealed additional minor proteins including caleosin, steroleosin, aquaporin and curcin. Information from these studies is essential for varietal improvement of Jatropha and/or developing oleosin gene fusion technology for expression of novel compounds. In this study 623 nucleotides upstream of the JcOleosin3 gene have been isolated and characterized in transgenic Arabidopsis plant using β-glucuronidase (GUS) as the reporter gene (Chapter 4). Result showed that the JcOleosin3 promoter directs iii expression of the β-glucuronidase gene in seed and pollen but not in leaves, root, stem or flower. In addition, in transgenic Arabidopsis, the JcOleosin3 promoter drives GUS activity at the early seedling stage; this was expected based on earlier reports of expression of Jatropha oleosins. To understand the molecular basis of seed germination, proteomic studies were performed on resting and germinating seeds of J. curcas. Such studies using 2-DE and MALDI-TOF MS revealed the presence of patatin-like lipases in the germinating seeds but not in the resting seeds (Chapter 5). These proteins showed sequence similarity and conserved domains as found in the patatin lipases from Solanum tuberosum and Arabidopsis thaliana. Patatin lipases play a significant role in the mobilization and lipolysis of TAGs during the germination of oil seeds. Further, there is little knowledge on the triacylglycerides (TAGs) lipid cycling in J. curcas., Bioinformatic data-mining confirmed the TAGs cycling in J. curcas to include the expected stages of synthesis, storage and lipolysis of fatty acids. Based on the data-mining results for the presence of various genes for TAG storage, synthesis and utilization, a scheme is presented for TAG cycling in J. curcas. The scheme presented in this study provides essential information required for oil manipulation in J. curcas.EThOS - Electronic Theses Online ServiceRoyal Thai GovernmentGBUnited Kingdo

Entamoeba chiangraiensis n. sp. (Amoebozoa: Entamoebidae) isolated from the gut of Asian swamp eel (Monopterus albus) in northern Thailand.

The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30-50 µm in length and 9-13 µm in width. Observed cysts were uninucleate, ranging in size from 10 to 17.5 µm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis

Blastocystis One Health Approach in a Rural Community of Northern Thailand: Prevalence, Subtypes and Novel Transmission Routes

Blastocystis is the most commonly found eukaryote in the gut of humans and other animals. This protist is extremely heterogeneous genetically and is classified into 28 subtypes (STs) based on the small subunit ribosomal RNA (SSU rRNA) gene. Numerous studies exist on prevalence of the organism, which usually focus on either humans or animals or the environment, while only a handful investigates all three sources simultaneously. Consequently, understanding of Blastocystis transmission dynamics remains inadequate. Our aim was to explore Blastocystis under the One Health perspective using a rural community in northern Thailand as our study area. We surveyed human, other animal and environmental samples using both morphological and molecular approaches. Prevalence rates of Blastocystis were 73% in human hosts (n = 45), 100% in non-human hosts (n = 44) and 91% in environmental samples (n = 35). Overall, ten subtypes were identified (ST1, ST2, ST3, ST4 ST5, ST6, ST7, ST10, ST23, and ST26), eight of which were detected in humans (ST1, ST2, ST3, ST4, ST5, ST7, ST10, and ST23), three in other animals (ST6, ST7, and ST23), while seven (ST1, ST3, ST6, ST7, ST10, ST23, and ST26) were found in the environment. In our investigation of transmission dynamics, we assessed various groupings both at the household and community level. Given the overall high prevalence rate, transmission amongst humans and between animals and humans are not as frequent as expected with only two subtypes being shared. This raises questions on the role of the environment on transmission of Blastocystis. Water and soil comprise the main reservoirs of the various subtypes in this community. Five subtypes are shared between humans and the environment, while three overlap between the latter and animal hosts. We propose soil as a novel route of transmission, which should be considered in future investigations. This study provides a thorough One Health perspective on Blastocystis. Using this type of approach advances our understanding on occurrence, diversity, ecology and transmission dynamics of this poorly understood, yet frequent gut resident

Circulation and colonisation of Blastocystis subtypes in schoolchildren of various ethnicities in rural northern Thailand

is a protist of controversial pathogenicity inhabiting the gut of humans and other animals. Despite a century of intense study, understanding of the epidemiology of remains fragmentary. Here, we aimed to explore its prevalence, stability of colonisation and association with various factors in a rural elementary school in northern Thailand. One hundred and forty faecal samples were collected from 104 children at two time points (tp) 105 days apart. For tp2, samples were also obtained from 15 animals residing on campus and seven water locations. Prevalence in children was 67% at tp1 and 89% at tp2, 63% in chickens, 86% in pigs, and 57% in water. Ten STs were identified, two of which were shared between humans and animals, one between animals and water, and three between humans and water. Eighteen children (out of 36) carried the same ST over both time points, indicating stable colonisation. Presence of (or ST) was not associated with body mass index, ethnicity, birth delivery mode, or milk source as an infant. This study advances understanding of prevalence in an understudied age group, the role of the environment in transmission, and the ability of specific STs to stably colonise children

High diversity of Blastocystis subtypes isolated from asymptomatic adults living in Chiang Rai, Thailand

Blastocystis is a common and broadly distributed microbial eukaryote inhabiting the gut of humans and other animals. The genetic diversity of Blastocystis is extremely high comprising no less than 17 subtypes in mammals and birds. Nonetheless, little is known about the prevalence and distribution of Blastocystis subtypes colonising humans in Thailand. Molecular surveys of Blastocystis remain extremely limited and usually focus on the central, urban part of the country. To address this knowledge gap, we collected stool samples from a population of Thai adults (n=178) residing in Chiang Rai Province. The barcoding region of the small subunit ribosomal RNA was employed to screen for Blastocystis and identify the subtype. Forty-one stool samples (23%) were identified as Blastocystis positive. Six of the nine subtypes that colonise humans were detected with subtype (ST) three being the most common (68%), followed by ST1 (17%) and ST7 (7%). Comparison of subtype prevalence across Thailand using all publicly available sequences showed that subtype distribution differs among geographic regions in the country. ST1 was most commonly encountered in the central region of Thailand, while ST3 dominated in the more rural north and northeast regions. ST2 was absent in the northeast, while ST7 was not found in the center. Thus, this study shows that ST prevalence and distribution differs not only among countries, but also among geographic regions within a country. Potential explanations for these observations are discussed herewith

Urban Diets Linked to Gut Microbiome and Metabolome Alterations in Children: A Comparative Cross-Sectional Study in Thailand

Loss of traditional diets by food globalization may have adverse impact on the health of human being through the alteration of gut microbial ecosystem. To address this notion, we compared the gut microbiota of urban (n = 17) and rural (n = 28) school-aged children in Thailand in association with their dietary habits. Dietary records indicated that children living in urban Bangkok tended to consume modern high-fat diets, whereas children in rural Buriram tended to consume traditional vegetable-based diets. Sequencing of 16S rRNA genes amplified from stool samples showed that children in Bangkok have less Clostridiales and more Bacteroidales and Selenomonadales compared to children in Buriram and bacterial diversity is significantly less in Bangkok children than in Buriram children. In addition, fecal butyrate and propionate levels decreased in Bangkok children in association with changes in their gut microbial communities. Stool samples of these Thai children were classified into five metabolotypes (MTs) based on their metabolome profiles, each characterized by high concentrations of short and middle chain fatty acids (MT1, n = 17), amino acids (MT2, n = 7), arginine (MT3, n = 6), amino acids, and amines (MT5, n = 8), or an overall low level of metabolites (MT4, n = 4). MT1 and MT4 mainly consisted of samples from Buriram, and MT2 and MT3 mainly consisted of samples from Bangkok, whereas MT5 contained three samples from Bangkok and five from Buriram samples. According to the profiles of microbiota and diets, MT1 and MT2 are characteristic of children in Buriram and Bangkok, respectively. Predicted metagenomics indicated the underrepresentation in MT2 of eight genes involved in pathways of butyrate biosynthesis, notably including paths from glutamate as well as pyruvate. Taken together, this study shows the benefit of high-vegetable Thai traditional diets on gut microbiota and suggests that high-fat and less-vegetable urban dietary habits alter gut microbial communities in Thai children, which resulted in the reduction of colonic short chain fatty acid fermentation

Prototheca bovis, a unicellular achlorophyllous trebouxiophyte green alga in the healthy human intestine

Introduction: Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely. Hypothesis/Gap statement: There is an absence of detailed characterization of human Prototheca isolates. Aim: The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool. Methodology: Prototheca was isolated from fecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed. Results: Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead. Conclusion: The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations

Relationships between some Thai cultivars of pineapple (Ananas comosus) revealed by RAPD analysis

RAPD analysis of nine Thai pineapple cultivars, including 'Phulae', 'Sawee', 'Tradsithong', 'Phuket', 'Pattavia', 'Intrachitdang', 'Intrachitkhow', 'Petburi No.1', and 'Nanglae', showed that, of 40 arbitrary 10- mer primers, 17 primers gave 206 DNA fragments ranging from 510 to 4,700 bp. One hundred and forty-five (70.4%) of the amplified fragments were polymorphic. RAPD analysis using NTSYS-pc Version 2.01e also showed that the similarity coefficients among the cultivars were 0.643-0. 963. The dendrogram indicated that the cultivars were clustered into 3 groups, consistent with the morphological data. The first group, consisting of 'Phuket', 'Phulae', 'Tradsithong', 'Sawee', and 'Petburi No.1', had morphological characteristics of the Queen group, while those of the second ('Intrachitdang' and 'Intrachitkow') and the third ('Nanglae' and 'Pattavia') groups could be determined morphologically to be members of the Spanish and Cayenne groups, respectively. 'Intrachitdang' and 'Intrachitkow' have similarity coefficient of 0.963, while that of 'Phulae' and 'Phuket' is 0.950. These pairs of cultivars are probably the same cultivars. The morphological differences between them are probably caused by mutations, differences in environment and agricultural practices, or combinations of these factors

Siamese Fighting Fish (<i>Betta splendens</i> Regan) Gut Microbiota Associated with Age and Gender

The Siamese fighting fish (Betta splendens Regan) is a popular ornamental fish in Thailand. Growing research suggests that fish health is influenced by gut microbiota. Here, we investigated, for the first time, the differences in the gut microbiota profiles of healthy Siamese fighting fish during the young (8-week-old) and adult male and female (16-week-old and 24-week-old) life stages using 16S rRNA gene sequence analysis. The fish were raised in controlled water quality conditions and fed on Moina macrocopa. Profiling of gut microbial communities revealed significant differences in the overall bacterial profile between young and adult Siamese fighting fish. Bacterial diversity decreased in the 24-week-old adult fish. Proteobacteria was the most predominant bacterial phylum in the gut of both young and adult carnivorous betta fish, in which the abundance of its members varied with age. Plesiomonas was enriched in male fish aged 24 weeks, whereas Pseudomonas dominated the gut of 8-week-old fish. Moreover, differences in predicted functions of these Proteobacteria between the young and adult fish could be a key target for improving fish growth. These findings expand our understanding of the role of gut microbiota and its association with host factors

Blastocystis subtypes in raw vegetables from street markets in northern Thailand

Blastocystis is a stramenopile protist of controversial pathogenicity. The organism colonizes a broad range of vertebrate and invertebrate hosts. Blastocystis has also been found in the environment both in water and soil. Several routes of transmission have been proposed including human to human, animal to human, and via contaminated food and water. In recent years, the presence of Blastocystis in vegetables has started to be explored. However, most studies have focused on microscopic detection. Moreover, works of this type from Asia are barely available. Hence, the aim of this preliminary study was to examine the occurrence of Blastocystis in raw vegetables sold in markets in northern Thailand. Fresh produce (n = 20) commonly used in Thai cuisine (Lanna) was purchased from two street markets and screened for Blastocystis using qPCR. Blastocystis was detected in 45% of the samples with the dominant subtype being ST3. Produce growing underground, such as galangal, carrot, and beetroot, were positive for the organism suggesting soil or inadequately composted manure as the source of contamination. To our knowledge, our study is the first to perform subtyping of Blastocystis in vegetables. Our results hint toward fresh produce being a, as yet, not widely explored, transmission route of Blastocystis in the studied community. Looking forward, large-scale investigations on the prevalence of this and other organisms under the One Health umbrella should be undertaken