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The Anopheles gambiae species complex includes the most predominant malaria vectors in sub-Saharan Africa. How they locate oviposition sites is not fully known, but a greater understanding of this subject may lead to more effective monitoring and trapping of gravid females. This project investigated potential oviposition semiochemicals and the oviposition behaviour of An. Gambiae sensu stricto Giles. The volatile profiles of seven bacteria were analysed by gas-chromatography linked mass-spectroscopy (GC-MS) to determine their volatile profiles and behavioural assays to determine their effect on An. gambiae. Cage assays could not confirm attraction to bacterial solutions, but demonstrated repellence to 4-methylphenol at 1mg/ml. GC-EAG demonstrated a strong electrophysiological response to 4-methylphenol and 8 of the 9 bacterial chemicals tested gave at least 50% of the 4-methylphenol response. Direct observations of oviposition in a large arena showed that, compared to a control dish of 0.9% saline, 1mg/ml 4-methylphenol reduced the number of visits, proportion landing and visit duration, but did not completely deter oviposition. A choice between dishes of saline or 1mg/ml 4-methylphenol, showed the latter was highly repellent; a majority of females oviposited in the control, the number, proportion and duration of visits to treatment dishes were reduced and fewer eggs were laid per female. When 4-methylphenol was presented separately from water in porous sachets, the repellency was shown to be largely volatile based, having an effect on the direction of approach to dishes, but no deterrence of egg-laying in dishes near the sachets. Short range cues, possibly involving substrate contact/sampling, appear to mediate the final stages of oviposition site selection. These findings are discussed in the context of utilising a more holistic approach than previously used to study mosquito oviposition; oviposition is clearly not a single behaviour, but a complex chain of sensory inputs and responses by the gravid insect.

I have constructed a genomic gene library of EcoRI fragments from Xenopus borealis DNA cloned in lambda-gtWES. The library contains 425,000 phage with a mean insert si ~ of 6.1kb. I have screened this library for histone genes and for a family of repetitive DNA sequences. I have isolated one histone gene clone from the library (Xbhl). It is overabundant in the library, so no other histone gene clones are isolatable. The cloned DNA fragment is 8.55kb long and contains one copy of each histone gene in the order H4-H2A-H2B-HI-H3 H2A is transcribed in the opposite direction to the other four genes. There is a short repetitive element between H2A and H2B present in 5000 copies in the genome, and an inverted pair of larger elements 3' to H2B present in/BOOO copies. I have isolated clones containing homologues to the latter and characterised them and homologous genomic sequences: it is a member of a dispersed, diverse family of elements with some sequence homology to his tome mRNA and present in anurans and urodeles. Xbhl is a minor species in the genome, but is homologous to the major histone gene cluster in the H4-H2A-H2B gene region. Its overall sequence is only 1-2% divergent from three histone gene clones isolated from X.laevis, which shared an ancestor with X.borealis 8 million years ago. The H4, H2A, H1 and H3 genes of Xbhl direct the production of normal proteins when Xbhl is microinjected into oocytes. The H3 gene codes a protein 96% homologous to Calf H3,and contains codons conserved in all H3 genes sequenced. The HI gene codes for a protein identical in 16 out of 21 central amino acids to an HI from X.laevis and homologous to calf and salmon HI proteins. It is possible that the H2B gene is a pseudogene.

The need to protect aquatic organisms from the toxic potential of environmental contaminants with minimal use of animal testing has been highlighted by the introduction of a number of regulatory directives. This thesis proposes the potential of 3D cell culture models to ‘bridge the gap’ between the use of existing subcellular and in vivo systems for the testing of compounds for potential persistence, bioaccumulation and toxicity, improving the predictive power of in vitro screening procedures. In this project, a trout hepatocyte spheroid model was optimised and using metabolic, fluorescence, transport and gene expression assays, the utility of this system in such studies as a superior alternative to currently used in vitro models was demonstrated. Hepatocytes cultured as spheroids remained viable for up to 40 days, exhibiting significantly greater functional xenobiotic metabolism and transport, as well as expression of genes, compared to other in vitro systems. These features of spheroids present a profile more closely representative of that of the whole liver. Together with the potential advantages of screening highly persistent compounds that require long term incubation and possible use in the assessment of the toxicity of compounds exhibiting chronic effects, this system successfully enriches current in vitro testing strategies.

The enhancer trap line P[GAL4]307 was isolated on the basis of expression in the giant fibres. It has proved to be an elegant marker of giant fibres in adult Drosophila. The giant fibres (GFs) are part of a simple circuit in the fly's central nervous system (eNS) that mediate a light-off escape response involving a jump and wing beat. Detailed analysis of the adult expression pattern, using antibodies to the products of the genetic elements used in enhancer trapping, has revealed that all the major components of the escape circuit are clearly detectable. Subsequently, the development of this circuit has been traced using the same antibody staining techniques, revealing the timing of circuit establishment and the developmental profiles of each component neuron. To complete this profile the birthdating of the GFs has been attempted. The overall aim of this section of work was to map the circuit from the first cell division producing the major neurons in embryos, through establishment of the circuit in pupae to full development of the active circuit in adults. The second major section of work has been the cloning of genes from DNA surrounding the P-element insertion. An important facet of P-element design is the ability to use it to remove the genomic DNA from around the site of insertion. A probe created from a fragment of this genomic DNA has been used to screen a library. This yielded a eDNA that, in combination with subsequently isolated cDNAs, forms a contiguous stretch of DNA approximately 4Kb in length. A translation of the eDNA has revealed homology with proteins from a range of eukaryotes from yeast to humans. These may form a family of elongation initiation co-factors. On the basis of homology with an Arabidopsis thaliana gene argonaute, this new Drosophila gene has been named diomedes. The genomic region has also been sequenced, revealing another gene close to diomedes. This new gene, LD07701, appears to be full length, and is unusual because it may be a non-coding RNA. Both genes isolated have neural expression in embryos, larvae and pupae, although it appears neither is the gene whose expression is reflected by the enhancer trap pattern.

The results presented in this thesis concern two aspects of the biology of trypanosomatids. First, the process of antigenic variation in the African trypanosomes and second the structure and function of the kinetoplast DNA of trypanosomatids. Part I. Trypanosomes were cyclically transmitted by the insect vector, Glossina morsitans, and the expression of variable antigen types (VATs) in the metacyclic populations from the salivary glands and the first bloodstream populations in metacyclic initiated infections in mice were analysed. Tsetse flies were fed on the blood of mice containing any one of 5 VATs of Trypanosoma brucei of the ANTAR 1 serodeme. The VATS of the metacyclic trypanosomes subsequently detected in the flies' saliva probes vere investigated using monospecific antisera to AnTAK 1 VaTs in indirect immunofluorescence and trypanolysis reactions; these sera included 5 raised against AnTats 1.6, and 1.45, previously idnetified as components of the metacyclic population (M-VATs), and against the 5 VATs originally ingested by the flies. The percentage of metacyclics reacting with a particular M-VAT antiserum remained more or less constant (AnTat 1.6, 6.0-8.3% AnTat 1.30, 13.7-18.2%; AnTat 1.45, 2.0-8.0%), regardless of the age of the fly or the ingested VaT. AS these 3 VATS account for no more than one-third of the metacyclic population, the existence of at least one more VAT is envisaged. The ingested VAT could not be detected among the AnTAR 1 metacyclic trypanosomes. Metacyclic trypanosomes from the salivary glands of infected tsetse flies were also used to initiate infections in mice. Immunofluorescence and trypanolysis reactions employing 24 monospecific antisera were used to analyse the VATS present in the mice following cyclical transmission. Regardless of the VAT used to infect tsetse flies, the first VATS detectable in the bloodstream were those previously identified as M-VATS. These were present until at least 5 days after infection, at which time lytic antibodies against at least 2 of the M-VATs were detectable in the blood of infected mice. In mice immunosuppressed by X-irradiation the M-VATs were detectable in the bloodstream for longer periods, but the percentage of the population labelled with anti-metacyclic sera showed a decrease on day 5 as in non-irradiated animals. The VAT ingested by the tsetse was always detectable early during the first parasitaemia following cyclical transmission and was usually the first VAT detectable after the M-VATs. Neutralization of selected M-VATs before infecting mice resulted in elimination of the neutralized M-VAT from the first parasitaemia but had no effect on the expression of other VATs in the early infection. Part II. In studies on the structure and function of the kinetoplast DNA (KDNA) of trypanosomatids I have examined the KDNA structure and mitochondrial activity of two species of Herpetomonas and also a stock of T. brucei which has lost the ability to activate its mitochondrion during syringe passaging in laboratory rodents. The structure of the KDNA of Herpetomonas muscarum and Herpetomonas inrenoplastis was compared by electron microscopy, restriction endonuclease digestion and hydridization with cloned portions of the maxi-circle from T. brucei 427. The KDNA of both H. musearum and H.ingenoplasits has a buoyant density of 1 .69B g/cm3; however, the KDNA of H. ingenoplastis represents 31% of the total cellular DNA as compared with 8% for H. muscarum KDNA. The KDNA network of H. muscarum consists of thousands of mini-circles of 0.6 to 0.7 x 106 daltons and a few large circular molecules, maxi-circles, of 21 x 10 daltons. The mini-circles of H. muscarum show sequence heterogeneity while maxi-circles of H. muscarum have a unique nucleotide sequence. The KDNA of H. ingenoplastis completely lacks mini-circle size molecules and the network is composed 6 6 6 entirely of large circular molecules of 11 x 106, 15.5 x 106 and 24 x 106 daltons. The 11 x 106 and 15.5 x 106 dalton molecules show sequence heterogeneity and are the major component of the KDNA. Hybridization studies with cloned fragments of T. brucei maxi-circle suggest that the 24 X 106 dalton component of H. ingenoplastis kDNA is functionally equivalent to the maxi-circle of other trypanosomatids. It was concluded that the 11 x 106 and 15.5 x 106 dalton circles of H. in enoplastis are functionally similar to mini-circles of other tryoanosomatids and that the maxi-circles of H. ingenoplastis differ from those of T. brucei and H. muscarum in major nucleotide sequences. The structure and activity of the mitochondrion from H.ingenoplastis and H. muscarum have been studied by electron microscopy, respiration studies with different substrates and inhibitors, analysis of oligomycin- sensitive ATPase activity and low-temperature difference spectra of respiratory chain cytochromes. Certain differences in the two species can be correlated with alterations in the maxi-circle of H. ingenoplastis described in the preceding paper.

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemical activities were found to coincide with oGSTs: TDRl was capable of reducing pentavalent arsenical and antimonial compounds to trivalent species, and possessed thioltransferase and dehydroascorbate reductase activities usually associated with glutaredoxins. TDRl, which was shown to probably reside in the parasite cytosol but may also be secreted, was found to be more abundant in amastigote than promastigote forms, which correlates with the antileishmanial stage-specificity of pentavalent antimonials. L. major TDRl knockout mutants were generated, and the protein was also over-expressed in parasites. Both these genetic manipulations resulted in mutants with enhanced infectivity. TDRl knockout parasites were more susceptible than wild type parasites to paraquat, which induces the production of intracellular superoxide. As its glutaredoxin-like in vitro activities suggest, this implies TDRl has a role in protecting the parasites from oxidative stress, although re-expression of TDRl did not reinstate resistance.

The development of axenic in vitro growth models of the human malaria parasite Plasmodium falciparum, has been pivotal in accelerating knowledge of this very important human pathogen. Despite the importance of this pathogen, there have been very few studies relating to the metabolism of the parasite. Furthermore, much of the preceding studies have been undertaken using culture conditions that do not accurately represent the physiological environment of the human host. There is a need to address whether different nutrient environments would trigger a parasite response at the systems level promoting a metabolic rewiring that would have an effect in progeny generation or life cycle progression. Because of its robustness, reproducibility and suitability for footprinting studies, NMR spectroscopy was chosen as the analytic technique for the study. One of the disadvantages of NMR is limited availability of software for identification and quantification of metabolites. This was taken as an opportunity to develop a pipeline using free, open-source programming framework. This tool was used to find unique and discriminatory metabolic profiles for both uninfected and P. falciparum infected red blood cells at various life-cycle stages using cell extracts and extracellular material. With the aim of studying parasite development in physiological conditions a culture medium mimicking human blood conditions was developed and tested on P. falciparum infected RBCs finding both phenotypic and metabolic differences. Further studies consisted of the development of tightly synchronised parasite cultures that were followed during 54 h using NMR-based metabolomics to assess consumption and excretion of metabolites in media, and high content imaging and bright field microscopy to assess parasite size and progeny. The measurements were taken under three different nutritional conditions: usual in vitro, physiological-like and hypoglycaemic. In usual culturing conditions P. falciparum 3D7 life cycle lasted around 45 h. During the early stages there was moderate consumption of glucose and glutamine and excretion of lactate, alanine and glycerol. During the mature trophozoite stages and schizonts, glucose uptake dramatically increased with a consequent augmentation of the lactate, alanine and glycerol production. These were excreted but their function was not clear. It was observed that these “wasteful" products were proportionally lower in the early developmental stages than in the later ones, suggesting a higher demand of raw materials (glucose) for biomass production during the early stages. During the late trophozoite stage the most abundant amino acids in the haemoglobin chain (leucine, valine and glycine) were excreted, a likely consequence of the need for space to nish maturation. Myoinositol, which is essential for creation of membranes was also avidly consumed. When comparing these findings with parasites growing in more physiological conditions there was a noticeable delay in the life cycle of at least 9 h. Consequently haemoglobin digestive products were excreted later in the time course. A decrease in the progeny resulting from schizonts containing significantly fewer merozoites was also observed. Parasites growing in physiological conditions but challenged with lower glucose availability also presented a further delay of the life-cycle and a decreased number of merozoites with respect to usual laboratory conditions. Haemoglobin degradation products were also excreted later in the life cycle and at lower rates compared to the parasites grown in complete media. These results suggest that there are significant differences between in vivo and in vitro life-cycles of P. falciparum. Such effects as the reduction in growth rates and elongation of the life cycle, if not accounted for, could severely compromise the in vivo results of in vitro drug killing rates assays.

Coral reefs are immensely vulnerable to climate change and particularly the effects of ocean warming; in efforts to understand whether and how reef systems will survive into the future, research is increasingly focusing on present day populations acclimatized to thrive under relatively extreme conditions. Whilst corals thrive along a range of environmental conditions, including relative extremes, within the Red Sea, these coral populations are still considered not well explored of the genetic and physiological signatures throughout this system. Corals microbiota communities (the “microbiome”) are recognized as a major component as to how corals “acclimatize” to different environmental conditions; therefore, this work aimed to investigate the historical thermal variability along the Red Sea and subsequently identify the relative role of coral microbiome associated with differences in coral thermal tolerance. Remotely sensed data (1982-2012) demonstrated migration of Sea Surface Temperature anomalies (i.e. DHW) from the south to the north during this time frame. Analysis of historical bleaching records indicated that coral populations were more tolerant to bleaching in the northern compared to the central/southern Red Sea. Symbiodinium clade type (ITS2) and microbial community (16S rRNA metagenomics) associated with six key coral species persisting across five sites of the northern Red Sea (29°-20°N) were then examined. Symbiodinium clade identity associated with each coral species generally remained highly conserved throughout the sites sampled. In contrast, microbial communities were variable within and between species across the Red Sea sites. Corals from two sites (central-Jeddah and northern-Hurghada) were exposed to a thermal stress experiment which confirmed that corals were more heat resistant at Hurghada (summer SST mean is 3.3 °C less) than Jeddah; however, symbiont ITS2 clade types were the same at both sites. Conversely, microbial community changed in heat stressed samples at Jeddah compared to the control group, while it remained stable at Hurghada. This work provides for the first time genetic analysis on corals’ microbiome inhabiting extreme thermal resistant region (i.e. the northern Red Sea) that contradict the global bleaching pattern. Our findings suggest that plasticity of microbial community may play the key role in acclimation of corals experience thermal anomalies in the Red Sea suggesting presence certain microbial phylotypes fill specific thermal niche. Finally, the higher latitudes of the Red Sea will broadly serve as a potential corals refugia which highlights the importance to conserve and implement a regional management policy to improve corals thermal tolerance of this region to be used as a genetic reservoir.

Taxonomic diversity, biology and ecological aspects of fungal endophytes of Tectona grandis (teak) from Chiang Mai Province, Thailand were investigated. It was found that the endophyte assemblages of mature leaves sampled from natural forest and plantation teak were not significantly different. Members of the Xylariaceae, especially Daldinia eschscholzii, Nemania subannulata, Hypoxylon haematostroma and Xylaria cubensis were frequent isolates. Widely reported endophytic fungi such as Phomopsis, Colletotrichum, Cladosporium and Fusarium were also isolated. There is little evidence to support host specificity for the majority of the isolates. Differences in endophyte assemblages between young and mature leaves were shown to occur with a much lower infection percentage in the young leaves. Species of Phomopsis and Colletotrichum were dominant in the young leaves but members of the Xylariaceae dominated in mature leaves. This pattern was the same for both natural forest and plantation samples. However comparison of taxa isolated from leaf lamina, midrib and veins gave conflicting results. Samples from mature leaves from natural forest trees exhibited little variation with greater variation in taxa recovered being found to occur between sampling years than between position of isolation from the leaf. In plantation leaves, although the results were similar to those from natural forest tree leaves for two of the years sampled, in 1997 the overall recovery rate was highest for the lamina, followed by veins and then the midrib. There was no evidence obtained to link individual taxa with specific regions of the leaf. It is now possible to devise a sampling strategy to obtain suitable diversity of endophytic isolates from teak leaves for industrial screening of these fungi. Techniques were developed to overcome current problems of identification of xylariaceous endophytes in the absence of their teleomorph. Inoculation of suitable woody substrata combined with selective incubation was used to induce teleomorph formation in many of the isolates and this together with chemical profiling enabled identification to species of many of these isolates. Rates of development of specific species were obtained and differences in environmental conditions necessary for development of teleomorphs to maturity were noted for members of different genera. Thus species of Daldinia and Hypoxylon required drier conditions than species of Xylaria and Nemania which only developed under wet shaded conditions. Xylariaceae from the natural forest, plantation, and forest surrounding the plantation were surveyed and a number of the Xylariaceae recovered as endophytes were found to be new to science, new records for Thailand or were recorded as endophytes for the first time.