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Thesis (Ph. D.)--Open University. BLDSC no. DX91237.

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Principal objectives of my research were to: (1) report for the first time that coelomocytes are able to reduce NBT dye and confirm the presence of lysozyme-like activity in earthworm; (2) develop a standard methodology for determination of NBT reduction and lysozyme-like activity in earthworms; (3) compare NBT reduction and lysozyme-like activity in earthworms with those of murine and human cells and fluids; and (4) demonstrate the sensitivity of earthworm NBT reduction and lysozyme-like activity as the assays using matrics in refuse-derived fuel fly ash (RDFF) and CuSO4.

The earthworm has long been of interest to biologists, most notably Charles Darwin, who was the first to reveal their true role as eco-engineers of the soil. However, to fully understand an animal one needs to combine observational data with the fundamental building blocks of life, DNA. For many years, sequencing a genome was an incredibly costly and time-consuming process. Recent advances in sequencing technology have led to high quality, high throughput data being available at low cost. Although this provides large amounts of sequence data, the bioinformatics knowledge required to assemble and annotate these new data are still in their infancy. This bottleneck is slowly opening up, and with it come the first glimpses into the new and exciting biology of many new species. This thesis provides the first high quality draft genome assembly and annotation of an earthworm, Lumbricus rubellus. The assembly process and resulting data highlight the complexity of assembling a eukaryotic genome using short read data. To improve assembly, a novel approach was created utilising transcripts to scaffold the genome (https://github.com/elswob/SCUBAT). The annotation of the assembly provides the draft of the complete proteome, which is also supported by the first RNA-Seq generated transcriptome. These annotations have enabled detailed analysis of the protein coding genes including comparative analysis with two other annelids (a leech and a polychaete worm) and a symbiont (Verminephrobacter). This analysis identified four key areas which appear to be either highly enhanced or unique to L. rubellus. Three of these may be related to the unique environment from which the sequenced worms originated and add to the mounting evidence for the use of earthworms as bioindicators of soil quality. All data is stored in relational databases and available to search and browse via a website at www.earthworms.org. It is hoped that this genome will provide a springboard for many future investigations into the earthworm and continue research into this wonderful animal.

Earthworms, Lumbricus terrestris, were challenged orally and intracoelomically with two bacterial species, Aeromonas hydrophila and Pseudomonas aeruginosa, and mortality rates were observed. Neither were found to be particularly pathogenic at injected doses of up to 108 bacteria per earthworm. The influence of Cu++ (as CuSO4) on the earthworm's response to bacterial challenge was investigated by exposing earthworms to sublethal levels of Cu++ prior to bacterial challenge. Exposure at sublethal concentrations up to 3 m g/cm2 did not have a pronounced influence on host resistance to challenge as measured by earthworm mortality. Cu++ increased the earthworm's ability to agglutinate rabbit erythrocytes, indicating that Cu++ exposure caused coelomocyte death, autolysis and release of agglutinins into the coelom, possibly explaining resistance to bacterial challenge.

Master of Science / Department of Agronomy / Ganga M. Hettiarachchi / Mine waste materials from the Tri-State mining region in Kansas, Missouri, and Oklahoma pose environmental hazards. The area is contaminated with trace elements, such as Pb, Zn and Cd, which are transported to surrounding areas through water, wind erosion, and runoff. Phytostabilization or establishing healthy vegetative cover could be used to reduce or control these contaminated materials from further spreading with wind and water. However, further research is needed to monitor the long-term sustainability and assess if high applications of compost amendments could help to facilitate soil reclamation. The overall focus of this thesis was to monitor long-term effects of compost or lime additions at two different rates, with or without other soil amendments, on soil properties, plants, and soil biota. We used the earthworm avoidance test as a screening tool for testing effects of soil amendments on ecotoxicity. In the first field study, pelletized manure compost additions of 448 Mg ha⁻¹ significantly decreased the bioavailable Pb, Zn, and Cd while increasing plant nutrients, vegetative cover, and plant biomass as compared to the contaminated control and the low addition of compost (224 Mg ha⁻¹) over 2.5 years. Plant tissue metal concentrations with compost addition did not show any phytotoxicity in this study. Lime additions did not show any significant effect on any of the measurements. Results from the first study suggest that one time addition of large quantities of compost at 224 to 448 Mg ha⁻¹ can support establishing and maintaining healthy vegetative cover at least for a 2.5 year period. In the second field study, long-term monitoring of the effectiveness of the amendments was studied. Compost was applied at two different rates (45 or 269 Mg ha⁻¹) in 2006. Various chemical properties, microbial activities, and vegetative growth or plant biomass were measured approximately for 4.5 years to evaluate long-term changes in soil quality and sustainability of phytostabilization efforts, when combined or assisted with soil amendments to improve the quality of trace element-contaminated mine waste materials. Plants grown with compost additions of 269 Mg ha⁻¹ showed higher nutrients, biomass, and enzyme activities as compared to plants grown on the contaminated control and with the low addition of compost (45 Mg ha⁻¹) over 4.5 years. Decrease in plant biomass and enzyme activities seen in the high compost treatments by the end of the study period suggested that long-term sustainability of these efforts may require repeated addition of soil amendments every 4 to 5 years. Additionally, a laboratory study was conducted to assess the long-term effects of treatments used in the second field study on ecotoxicity using the avoidance behavior responses of the earthworm Eisenia fetida. There was no mortality of earthworms after a 48 h exposure period of any of these treated and untreated mine waste materials. Avoidance was clear for the contaminated control and the low compost treatment (45 Mg ha⁻¹). Moreover, the contaminated control did show habitat limitation (< 20% of earthworms was found in test soil). Current research studies provide evidence that, high rates of compost applications can be used to stabilize and reduce the bioavailability of trace elements in mine waste materials.