Some strains of Lysinibacillus sphaericus bacteria produce proteins toxic to insects, in particular mosquitoes. The bacteria have been used on a large scale to control these disease vectors. Unfortunately, there have been some reports of insect resistance to L. sphaericus. Interestingly, there are also reports of new isolates that may provide an opportunity to exploit these bacteria in other ways. To achieve better control of insect vectors, studies of various aspects of the L. sphaericus and its toxins are needed. This dissertation reports properties of a newly isolated L. sphaericus isolate namely, Fang Large. Based on its morphological features and 16S RNA DNA sequence analysis, the Fang Large isolate was confirmed as a Lysinibacillus sphaericus strain. This isolate exerts its pathogenicity to Wax moth larvae through septicaemia. Next, regulation of toxin gene expression was performed through analysis of 5’ untranslated regions of L. sphaericus toxin genes of strain IAB59 using a 5’ RLM-RACE method to reveal the promoters of the toxin genes. These investigations did not always produce the expected results but 5’UTR analysis of the mtx2 sequence confirmed the previously predicted promoter, while for two other genes –binB and cry48– results indicated that the actual promoters are not those previously predicted and that unreported promoter sequences appear to act in regulating these genes. To study the genomic arrangement of L. sphaericus strain NHA15b and its toxin protein genes, next generation sequencing technology was employed. This showed that the chromosomal structure of strain NHA15b is similar to the previously sequenced L. sphaericus genomes, but lacked a 35 kb fragment that in strain C3-41 is known to harbour binA/B and mtx4 genes. There are at least five (possibly six) plasmids in L. sphaericus strain NHA15b. Two of them, pLsph100 and pLsph200 are similar to other plasmids reported from other L. sphaericus strains. Plasmids pLsph300, pLsph400 and pLsph500, in contrast, have never been reported in any L. sphaericus strain. The sixth plasmid, pLsph600 was predicted based on the presence of a T4SS system and DNA replication protein genes. The genes may be responsible for the conjugation ability of the particular plasmid. It is predicted that the cry48/49 genes of the strain NHA15b may be located in this putative plasmid. The prediction is supported by indications from physical separation efforts using Pulsed Field Gel Electrophoresis, that the cry genes were located in a plasmid with mobility equivalent to 750 kb linear DNA. This prediction still needs further verification.

A previous study showed that unlike X. laevis, X. borealis contains a predominant or major histone gene cluster, which contains 70% of the 80-90 copies of the H4 genes in the genome (Turner & Woodland 1983 (Nucl. Acids Res.11 978)). This prompted more detailed analysis of histone gene arrangements in X. borealis, which is the subject of this thesis. Clones containing histone genes were isolated from a library prepared for this purpose. Analysis of these clones indicated one class containing the major cluster, and a second, minor class which appeared to be cloned at high frequency. Representative members of each class were characterised in detail. Major cluster clone XbHW302 was restriction site mapped, and certain regions sequenced. Microinjection of the major cluster clones in to Xenopus oocytes, confirmed that these genes were functional. Attention was then turned to the chromosomal organisation of the major cluster. A 'chromosome walk' experiment allowed the isolation of clones indicating a tandem of arrangement of clusters on the chromosome. This allowed the complete 'repeat' to be mapped in detail, further studies confirmed that the majority of clusters are tandemly repeated in the genome. The minor cluster clones were analysed in a fashion similar to the major class. Restriction site mapping and DMA sequencing of clone XbHW61 allowed the location polarity, and identity of an H3, H4, and an HI gene to be determined. Microinjection analysis again showed the genes were functional. Extensive DNA sequence comparisons between various Xenopus histone gene clusters were undertaken. The picture of histone gene cluster structure in X. borealis that emerges from these studies, is compared at the nucleotide, gene, and chromosomal levels with X. laevis. A discussion of how the differences between such closely related species could have arisen is also included.

Fish are in contact with microbiota from the moment of hatching. Exterior organs, i.e. skin, gills and intestinal system, are colonised by commensal bacteria populations and a symbiotic relationship is formed. The fish provides a niche and nutrients for the bacteria which stimulate development of the immune response, act as an additional barrier against invading pathogens and, within the gut, aid in digestion. β-glucans are used within aquaculture as a means of improving fish health and can be applied in various forms, e.g. via diet or injection. Whilst the application of β-glucan is performed to modulate a fish’s immune system, it has also been shown to affect the gut microbiota population at concentrations above 1% w/w within the diet which is particularly important to consider when applied orally. The effect of the commercially available β-glucan MacroGard® upon the gut of common carp (Cyprinus carpio) is studied after oral application and injection. Whilst feeding with MacroGard® at 0.1% w/w within the diet does not influence the gut bacteria nor expression of bactericidal innate immune genes, injection (2mg kg-1 and 5mg kg-1) resulted in a 90% reduction in bacteria numbers in the gut after 24 hours. Injection of MacroGard® did not significantly alter the expression of CRP, iNOS, bf/C2, IL-1β, ApoA1, HAMP1, LEAP2 and Muc2 within the gut however MSS1, a synthesised β-glucan, significantly increased the gene expression of iNOS, CRP and Muc2. 0.1% MacroGard® in the diet was, however, capable of influencing bacterial species diversity when injection was also performed. This revealed a high proportion of Alphaproteobacteria, which are typically associated with plants rather than gut systems, and corresponded with a reduction in potential pathogenic bacteria. This showed combining injection and oral application of MacroGard® together is capable of influencing the gut microbiota population within a 2 week period.

No description available.

No description available.

Bats are vital to Great British biodiversity; they are the primary consumers of nocturnal insects, disperse nutrients across landscapes, and are excellent bioindicators of an ecosystem’s health. The diversity of bat species in the UK is thought to be as a result of extensive resource partitioning. There are numerous methods used for studying bat diets, each with benefits and drawbacks. Past research has compared small numbers of species at a time, making inter-species comparisons difficult. Our large repository of bat guano samples, collected from around the UK, has allowed us to study the bat species under one methodological ‘umbrella’. This thesis is divided into 7 chapters. This first chapter gives a broad overview of the project, framing this research and provides an overview of the technologies available, and how their development has enabled environmental research on a scale, which, until recently, would have been unimaginable. The second chapter is a meta-analysis of the literature that pertains to bat diets. These data will be used to inform the design of primers in the barcoding stages of the project. Next is a shotgun metagenomic analysis of a selection of guano samples from across the range of the UK species. This method provides information, not only about diet species, but also about the bat, viral, and bacterial DNA. Analyses of this data show that there are several dietary forms seen between the species. The fourth chapter is a targeted amplicons metagenome study of the mitochondrial COI barcode region from the arthropod species identified in the literature review, and from metagenomic data-set. This provides a greater resolution picture of the diet species present. Analyses of these data use phylogenetic intersection analysis to ensure the robustness of the taxonomic assignments in the face of the patchy databases available. In the fifth chapter, I draw together the data gathered using the different approaches and presented in the previous chapters. I discuss the efficacy of the methods, and assess the role of resource partitioning in bat species co-existence. The sixth section will look at the appropriateness of using guano morphology as a diagnostic of species presence. Finally, in chapter seven, I summarise these data in the wider context of bat ecology, comment on the implications of the research for conservation, and discuss potential directions for the field in the light of this research.

Maternal effects are ubiquitous in free-living organisms, with parent-environment interactions affecting offspring life-history traits and fitness. These effects have been demonstrated in a wide variety of organisms, including mammals, insects and plants. Despite this, there is little evidence of maternal effects existing in parasites. If maternal effects are so prevalent in free-living organisms then it is unlikely that they do not exist in parasites. Maternal effects are important because they influence progeny fitness, measured by fecundity, longevity and developmental time. If they exist in parasite then they likely result in increased virulence, greater persistence (especially in terms of soil dwelling nematodes) and may be a driving influence in selection for resistance. Here, maternal effects are demonstrated in the free-living nematode Caenorhabditis elegans and the maternal effect of temperature demonstrated under constant and variable conditions. Similarly, the fitness effects of environmental temperature are explored in entomopathogenic nematodes and the effects of both maternal temperature and host species are demonstrated to cause changes in offspring development. Finally, C. elegans is used to demonstrate the importance of maternal effects in parasite life history and their potential impact on parasite control. The implications of altered life-history strategies that come about as a result of mothers tailoring their reproductive strategies in response to environmental cues for agricultural and medical parasite control are discussed. Critically, the effects of low parental exposure to anthelmintic compounds and nematicidal plant extracts on the fitness of offspring are demonstrated. This work provides evidence for the existence of maternal effects in freeliving and parasitic species and highlights the importance of the recognition of such effects in multiple fields.

The metabolism of the mammalian form of Leishmania mexicana mexicana has been investigated by comparing the amastigote activities of a number of enzymes involved in the glycolytic sequence, tricarboxylic acid cycle and associated pathways with those of the promastigote. Initial studies suggested that there are only quantitive differences between the two forms with the greatest of the differences found in the area of CO2-fixation. Phosphoenolpyruvate carboxykinase (PEP carboxykinase) was detected at much higher activity in amastigotes than promastigotes. This enzyme was found to be ADP specific and to have an absolute requirement for Mn2+. There appeared to be no regulation of the amastigote enzyme in crude homogenates by ATP, GTP, ITP or the end products malate and succinate. Pyruvate carboxylase was undetectable in either parasite form and malic enzyme (carboxylating) was detected only at low activity in promastigotes. Amastigotes had low pyruvate kinase but high malate dehydrogenase activities in comparison to promastigotes. This suggests that in amastigotes the main catabolic route for PEP is to succinate, a major end-product of metabolism, by way of carboxylation and reduction involving malate dehydrogenase and fumarate reductase. In contrast, the high activity of pyruvate kinase in promastigotes suggests that, in this parasite form, PEP is more likely to be converted to pyruvate which either enters the TCA cycle or is transaminated to alanine. Promastigotes had a high NADH-linked glutamate dehydrogenase activity in comparison to amastigotes and may reflect the higher glutamate utilization by the insect form of the parasite. A low NADPH-linked glutamate dehydrogenase activity was found in amastigotes, whereas none was detected in promastigotes. Isocitrate lyase could not be detected in either form, suggesting that the glyoxylate cycle plays no part in L. m. mexicana metabolism. The theory that the glycolytic kinases, and in particular phosphofructokinase, are primary targets in the action of pentavalent antimonial drugs against Leishmania was investigated with L. m. mexicana. Pentostam (sodium stibogluconate), active in vivo against Leishmania, had no effect on promastigote growth in vitro even at concentrations of 100μg/ml. In contrast, Triostam, the trivalent analogue of Pentostam, inhibited the growth in vitro of L. m. mexicana promastigotes with an LD50 of 20μg/ml and an MLC of 400μg/ml. It was found to be almost as effective on a weight/weight basis as the trivalent arsenical melarsen oxide (LD50, 20μg/ml; MLC, 100μg/ml). The leishmanicidal effect of Triostam, however, could not be correlated with any enzyme inhibitory activity. Neither Pentostam (110μM) nor Triostam (600μM)inhibited hexokinase, phosphofructokinase, pyruvate kinase, malate dehydrogenase or PEP carboxykinase from either amastigote or promastigote. In contrast, melarsen oxide was a potent inhibitor of all leishmanial enzymes tested except hexokinase. The action of Triostam was antagonised by cysteine indicating that the drug's action may involve thiol groups in some way. Triostam, but not Pentostam, inhibited phosphofructokinase of adult Schistosoma mansoni with an I50 of 200μM, suggesting that antimonials may act in different ways against schistosomes and Leishmania. The subcellular organisation of L. m. mexicana promastigotes was investigated using differential and isopycnic centrifugation. Glycosomes and mitochondrial vesicles from culture promastigotes were separated on linear sucrose gradients. Hexokinase, glucose phosphate isomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and PEP carboxykinase were recovered largely in association with glycosomes (density; 1.215 g/ml). Phosphoglycerate kinase and glucose-6-phosphate dehydrogenase had some glycosomal activity but were mostly recovered in the soluble fractions. Pyruvate kinase was totally cytosolic. Malate dehydrogenase showed a broad peak corresponding to that of the mitochondrial marker oligomycinsensitive ATPase (density; 1.190g/ml). Glutamate dehydrogenase and alanine aminotransferase both showed small mitochondrial peaks, but most of the activities were recovered elsewhere on the gradient and in the soluble fractions. Amastigotes of L. m. mexicana were not successfully fractionated using the technique developed for promastigotes, therefore the subcellular location of enzymes in amastigotes was investigated by following the release of soluble enzymes from digitonin-treated amastigotes. This revealed distinct cytosolic, mitochondrial and glycosomal compartments. The findings give an insight into the organisation of L. m. mexicana promastigote and amastigote energy metabolism. The importance of PEP carboxykinase and malate dehydrogenase to the metabolism of the amastigote prompted a more detailed investigation of these two enzymes. Isoelectric focusing studies showed that amastigotes possessed particulate malate dehydrogenase isoenzymes apparently absent from promastigotes. The particulate activities of amastigote malate dehydrogenase and PEP carboxykinase were purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5' AMP-Sepharose 4B and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. PEP carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SOS gel electrophoresis were 39,8OO and 33,300 for malate dehydrogenase and 63,100 and 65,100 for PEP carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km NADH of 41μM and a Km oxaloacetic acid of 39μM. For malate oxidation there was a Km malate of 3.6mM and a Km NAD+ of 0.79mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than O.83mM and malate was found to be a product inhibitor at high concentrations, however there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts, nevertheless their apparent importance and unique subcellular organisation in the parasite make them potential targets for chemotherapeutic attack.

Carrion represents an important energy resource in the natural world, yet its ecological significance has often been overlooked. It also plays a crucial role facilitating energy transfer across trophic levels and between ecosystems. The aim of this thesis was to evaluate the role of marine mammal carrion in the ecology of coastal systems, investigating one of the most commonly occurring sources of marine mammal carrion in the UK, the grey seal (Halichoerus grypus). This was addressed by providing first a detailed documentation of the fate of a grey seal pup carcass with insights on the scavenging ecology existing in both terrestrial and marine ecosystems. On the shore, great black-backed gulls (Larus marinus), juvenile gulls and ravens (Corvus corax) fed on the carcass showing a distinct temporal succession using the food resource. The underwater carcass was initially dominated by Echinodermata (starfish), whose abundance dropped lately, while Malacostraca (crabs) were present in similar number during the whole monitoring. Bacterial activity was evident in both experiments. Predictability of seal carrion was then defined during the pupping season at one of the largest colonies in the UK, the Isle of May (Scotland). Data collected by aerial survey (11 years) and ground visual census (3 years) were used to estimate the inter-annual variability of carrion, from placentae and dead seals, according to its timing, biomass and energy released and spatial distribution on the island. For all measures considered, variability was below 34%, similarly to other resources, such as salmon runs, which appear to be predicted by consumers. Twenty one percent of the total biomass from dead seals was consumed at the end of the pupping season suggesting a clear response from the scavenging community to the presence of the resource carrion. The predictable nature of seal carrion was then tested exploring the spatial and temporal distribution of scavenging gulls at three geographical scales (regional, local and patch scales). The great black-backed gull was affected by carrion availability occurring on the Isle of May, while the herring gull (Larus argentatus) was not. In particular, the number of adult and juvenile great black-backed gulls feeding was directly correlated with carrion abundance, while searching behaviour was greatest after the mean seal pupping date and at the peak in mortality. The behavioural dynamics of scavengers were finally explored monitoring the feeding activity on pup carcasses and placentae of adult and juvenile great black-backed gulls. It was predicted that under conditions of predictable and abundant carrion an equal scavenging effort would be found for the two age classes when consuming carcasses. Hierarchical dominance was, instead, expected during scavenging activity on placenta as it represents a preferred energy-rich food item. Temporal trends of scavenging activity and time spent feeding on carcasses were in fact similar between the two, while young individuals spent more time feeding on placenta, highlighting the importance of this food source for juvenile gulls during winter. The house mouse was also found to scavenge on seal carrion, which until now has been undocumented. This study demonstrates the importance of marine mammal carrion as a resource for multiple facultative scavenger species in both the marine and terrestrial environment.

Crayfish are keystone species and ecosystem engineers that affect the structure and function of aquatic ecosystems. Whilst ecological impacts are caused by crayfish in their native range, non - native crayfish species typically have a greater ef fect on some other aquatic organisms and ecosystem processes (Chapter 2). Crayfish are extremely successful invaders that often cause declines in native cra yfish (Chapter 3). Of the 7 non - native crayfish species in the UK, the signal crayfish ( Pacifastacus leniusculus ) is currently the most widespread (Chapter 3). Field and laboratory data, however, suggest that in parts of the UK signal crayfish are being outcompeted by more recently introduced virile crayfish ( Orconectes cf. virilis ) (Chapter 4). Non - native crayfish also threaten native crayfish through disease, notably crayfish plague ( Aphanomyces astaci ), transmission. Whilst non - native North American crayfish are largely resistant to A. astaci , infection in susceptible native European species is usually lethal. Within this study 23 signal crayfish populations were screened for A. astaci and 13 were infected (Chapter 5). Virile crayf ish from the UK were also infected with A. astaci , and therefore should also be considered as a transmission pathway for this pathogen in the UK (Chapter 6). Whilst the majority of studies on crayfish symbionts are focused on A. astaci , crayfish host a wid e range of micro and macro - parasites. One group of particular interest are branchiobdellidans (Annelida: Clitellata). Two species of these ectosymbionts, Xironogiton victoriensis and Cambarincola aff. okadai , were recently discovered on invasive signal cra yfish in the UK (Chapter 7). Owing to their abilities to survive for extended periods off the host and reproduce rapidly both species have a high invasion potential in the UK (Chapter 8). Laboratory experiments show that signal crayfish infested with X. victoriensis were less aggressive and poorer foragers than uninfested c rayfish , therefore these symbionts may influence signal crayfish invasion dynamics (Chapter 9).