No description available.

No description available.

No description available.

This is a study on the diversity of entomopathogenic nematodes (EPN) in Chile and the bionomics of a new EPN species, Steinernema australe. Surveys for EPN were carried out in six regions of Chile, representing some of the principal habitats of the country and including desert, sub-polar, montane and rainforest habitats, by collecting approximately 1400 soil samples. Live-baiting of the samples revealed 101 isolates, 94 of which were Steinernema and seven were Heterorhabditis. Morphological and molecular characterisation revealed one species of Heterorhabditis, designated as Heterorhabditis sp. 1 and three species of Steinernema, viz., S. feltiae and two previously undescribed species, S. australe and S. unicornum. Heterorhabditis sp. 1 was only found in the northern half of the country, the majority of Steinernema were found in the southern half. The most southerly survey, Patagonia and Tierra del Fuego, had the highest proportion of positive soil samples at 18.3%, made up of S. feltiae and S. unicornum; surveys in the Atacama Desert had the lowest return (1-2%), consisting only of Heterorhabditis sp. 1. There were indications of molecular and geographical intraspecific variation in S. feltiae and S. unicornum. The incidence of the four species and a summary of habitat characteristics are provided, as well as taxonomic descriptions of S. australe, S. unicornum and Heterorhabditis sp. 1. Profiling of S. australe revealed a fast life- cycle, with new infective juveniles (IJ) observed after 6 days at 20°C and significant infection at cool, humid conditions. Five days at ea -1°C had no effect on IJ survival, but subsequent infectivity was reduced, a possible chilling-injury. Steinernema australe infected a wide range of insect pests, appearing most effective against Lepidoptera. No nictating or jumping behaviour was observed. Xenorhabdus sp. and Paenibacillus sp. of bacteria were recovered from S. australe.

No description available.

Longevity and the rate of senescence are determined by the ecological conditions during a population's recent evolutionary history, and are intrinsically linked to other components of life history and to fitness. These traits should be examined in an ecological context, in which other aspects of the life history are taken into account. However, although many mutations which promote longevity in model organisms disrupt mechanisms that are involved in responding to environmental change, trade-offs associated with increased lifespan have typically been examined in benign laboratory conditions. In the nematode Caenorhabditis elegans, long-lived, stress resisitant age-1(hx546) mutants can compete with wild type worms in favourable growth conditions, but display fitness costs when populations are periodically starved. By monitoring termporal changes in genotype frequencies, I have established that age-1 mutants can have higher fitness that the wild type strain if mixed genotype populations are exposed to periods of thermal or oxidative stress when food is available. Genotype-by-environment interactions, and spatial and temporal distributions of the FOXO transcriptions factor DAF-16, suggest that this is because age-1 mutants are more able to survive, develop and reproduce during and/or after exposure to environmental stress, due to increased expression of genes involved in somatic maintenance and repair. Using population projection matrices, I have demonstrated that age-1(hx546) mutant allele can confer a selective advantage over the wild type genotype when populations experience abiotic stress, even if periods of starvation are frequently endured. This is the first demonstration that a long-lived, laboratory-derived mutant can have higher fitness that a wild type genotype under specific environmental conditions. The results imply that, is genetic variation is present in populations which encounter harsh conditions, increased longevity can evolve as a consequence of selection for greater resistance to stress. I have also established that the effects of mutations which promote longevity on the ability to tolerate environmental stress can be context dependent and that long-lived age-1(hx546) mutants display increased cold tolerance, relative to wild type worms, due to increased expression of ∆9 desaturase genes and additional transcriptional targets of DAF-16. The results presented in this theis suggests that genetic and life history responses to environmental stress deserve a more prominent role in evolutionary studies of aging.

Caenorhabditis elegans, a millimeter-sized, soil-dwelling nematode, is a model organism for biology research. Its whole genome has been sequenced. The lineage and fate, for each one of the cells in wild-type (N2) worms, is known. The connectivity, for all 302 neurons of wild-type hermaphrodites, has been mapped. Many of its genes have homologs within other organisms, including humans. C. elegans have a well-defined repertoire of observed behaviors. For these reasons, and due to a wealth of experimental data, C. elegans is a well-suited organism for mapping genetics to phenotype. This thesis details a system for relating genetics to phenotype. I present a methodology for semi-automated, high-throughput, high resolution investigation of gene effects on behavior and morphology using C. elegans. In the first section beyond the introduction, Chapter 2, I describe a new singleworm tracking system (hardware and software), titled Worm Tracker 2.0 (WT2), which was used to collect videos of worm behavior with high throughput. While multi-worm tracking systems exist, including ones that enable higher experimental throughput by recording multiple worms at once, their videos have insufficient resolution to resolve worm bodies well and these systems have been limited to only simple measurements. While other single-worm tracking systems also exist, they present, among other limitations, significant costs precluding high experimental throughput. I designed and built the hardware and software for a less expensive unit, which is approximately 1/4 the cost of previous single-worm trackers. This enabled us to purchase eight such units for high-throughput of experimentation. Other novelty for our system includes the ability to track worms at all larval stages and the ability to follow single-worms swimming. In Chapter 3, I describe a novel automated analysis for the worm videos collected using the aforementioned single-worm tracker. While analysis exists for other single-worm tracking systems, several limitations precluded adaptation. Our worm videos are on food and the worms are of variable size. Several previous algorithms attempted to deal with worms on food but, for our purposes, suffer from poor resolution at the head and tail, areas necessary to obtain significant phenotypic information. The analysis I built uses a novel algorithm driven by a need to obtain high-accuracy and precise worm contours (and their consequent skeletons) in our difficult conditions (e.g., on food and swimming environments) with invariance to worm size (bounded by a minimal limit of resolution). This accuracy was necessary due to the sheer size of the data set collected, roughly 1/3 of a billion frames, which precludes manual verification. In the final section, Chapter 4, I describe the results from my analysis of our collected data. Using our trackers we collected more than 12,000 videos, each 15 minutes in length, at 640x480 20-30Hz resolution, representing over 300 mutant strains matched to wild-type controls. This large set was filtered to obtain high-quality data and remove strains specific to private data sets (prepared for future publications). The filtered analysis covers 330 worm groups compiled from 300 mutant strains, 2 wild isolates, three descendants of N2, along with our N2 controls divided into hourly, daily, and monthly groups. A subset of 79 strains, representing 76 genes with no previously characterized phenotype, show significant measures in my analysis. Further sensitivity of the analysis is explored through measures of habituation, small morphological changes due to growth, and a phenotypic comparison of the three descendants from the ancestral, wild-type N2. With the sensitivity explored, I present an N2 phenotypic reference compiled from 1,218 worms, recorded over three years. Statistics of this set define a reference measure of the N2 phenotype (specific to the Schafer Lab wild type) with broad implications for performing and controlling C. elegans experiments. Three genes, implicated in mechanosensation as a result of genetic sequence but lacking any observed phenotypic support, reveal locomotory phenotypes in our analysis. This prompts a large clustering of all 330 groups, to assess the predictive capabilities of our system. The N2 groups cluster together in a large exclusive aggregate. Further support for the predictive capabilities of the clustering emerge among multiple published pathways that also form exclusive clusters. I end by discussing a set of genes, predicted to be acetylcholine receptors through genetic sequence and functional heterologous expression, which now receive further support through strong aggregation within their own exclusive phenotypic cluster.

The epithelial monolayer of cells surrounding the animal, the hypodermis, Synthesises five cuticles during the nematode life cycle. The first cuticle is formed within the egg, prior to hatching, and the remainder towards the end of each larval stage. Because of the structural role of the cuticle, mutations in genes involved in assembly of this ECM can cause a spectrum of effects from lethality late in embryogenesis to alterations in the nematode shape. The severity of phenotype correlates with the severity of cuticle synthesis defects. Accordingly, two distinct mutant alleles that cause death after embryonic elongation, possibly due to failure in synthesising an intact cuticle, were characterised . One mutant, ij15, was isolated from a forward genetic screen previously performed (I. Johnstone, Glasgow University, Glasgow, UK). ij15 defines mutationally the gene stc-1, which encodes a HSP70-like protein possibly localised in the secretory pathway. The other mutant, h402, defines mutationally the gene let-607. A second let-607 allele, h189, which results in larval lethaity at the L2 stage was also analysed in this study. let-607 corresponds to the predicted gene F57B10.1, which encodes a putative bZIP transcription factor. Both stc-1 and let-607 are expressed in the hypodermis at all developmental stages. Furthermore, disruption of the function of either stc-1 or let-607 by mutation or RNAi affects cuticle synthesis in different ways. Thus, stc-1 and let-607 encode for a HSP70-like protein and a putative bZIP transcription factor required for synthesis of the cuticular ECM in C. elegans. In addition, this study defines C. elegans mutant phenotypes that can be used as indicators for gene products with controlling roles in the synthesis of this ECM.

<i>Trichinella spiralis</i> is a parasitic nematode of mammalian skeletal muscle.  Its life cycle includes two stages where developmental progression appears to be inhibited until a specific host niche is encountered.  The newborn larva, released within the host intestine depends upon entry to skeletal muscle for continued development.  The muscle larva encapsulates within skeletal muscle and further reproductive development is dependent upon ingestion by a new host.  Developmental arrest has been extensively characterised in <i>Caenorhabditis</i> <i>elegans</i>, where an alternative L3 larva, the dauer larva, is formed in response to environmental conditions refractive to continued reproductive development.  Using the wealth of genetic information regarding <i>C. elegans</i> dauer formation, putative periods of arrest were examined in <i>T. spiralis.</i> TGF-<span lang=EN-GB style='font-family:Symbol'>b-like and insulin-like signalling pathways are critical mediators of <i>C. elegans </i>dauer formation.  A <i>T. spiralis</i> TGF-<span lang=EN-GB style='font-family: Symbol'>b-ligand was identified and designated <i>ts-tll-1</i>.  Sequencing and analysis revealed <i>ts-tll-1</i> to be similar to vertebrate bone morphogenetic proteins and <i>C. elegans </i>DBL-1, is involved in body size regulation.  EST mining identified putative type I and II TGF-<span lang=EN-GB style='font-family:Symbol'>b receptors and a subtilsin-like proprotein convertase, suggesting conservation of TGF-<span lang=EN-GB style='font-family:Symbol'>b-like signalling in <i>T. spiralis.  </i>A partial <i>Trichinella </i>gene encoding an orthologue of the <i>C. elegans</i> insulin-like, tyrosine kinase receptor, DAF-2, was identified by degenerate PCR and designated <i>ts-tkr.  ts-tkr</i> is most similar to <i>C. elegans daf-2</i> within the highly conserved tyrosine kinase domain.  Two alternative transcripts of <i>ts-tkr</i> were identified by 3’ RACE, which differed in their 3’ UTRs.  Semi-quantitative RT-PCR analysis suggested <i>ts-tkr </i>expression was greatest in adult worms, implying a role in promoting reproductive development. Semi-quantitative RT-PCR was also to assess the expression of selected housekeeping and ES protein encoding genes during the <i>T. spiralis </i>life cycle.  While transcription in the <i>C. elegans</i> dauer is depressed, there was no obvious transcriptional repression in <i>Trichinella</i> newborn or muscle larva.

Surveys for entomopathogenic bacteria and their associated nematode hosts were conducted locally (University of Bath campus) and across southern England. Sampling involved trialing a novel Android app. (Epicollect) to manage sample collection data. Galleria larvae were used to bait UK soil samples. Insects which became infected were placed on White traps to collect any emerging nematodes, from which bacteria were isolated. Bacteria were also isolated from the haemolymph of any infected larvae. Bacterial isolates were classified on the basis of 16s rDNA and recA gene sequences. Serratia proteamaculans-like strains dominated the samples, and Multilocus sequence analysis (MLSA) was developed for the characterization of these Serratia isolates. We determined the sequences of (350-450-bp) fragments from five housekeeping genes of 84 isolates of Serratia proteamaculans. MLSA was shown to be effective for distinguishing closely related strains found in the insects’ haemolymph and from different nematodes. goeBURST was used to visualize the relationships between the STs, and the data showed a high level of discrimination, resolving 69 STs from the 84 isolates. In addition, the data derived from this study were represented in a phylogenetic network using the Splits Tree-network methods, to show the rate of recombination within and between the genes. From a total of 256 infected Galleria 23.04% were nematode positive. The nematodes were identified based on 18S rDNA 19 isolates were close relatives of the species Pristionchus entomophaga and Diplogasteriodes magnus (Diplogastridae). A further 16 isolates were more closely related to Steinernema glaseri (Steinernematidae). All three nematode types were isolated from diverse habitats and soil types, but were isolated more frequently in cold seasonal conditions. The bacterial sequence data suggest that the nematode- associated strains of bacteria belong to specific clades, distinct from the free living infective strains, which hints at ecological diversity within the S. proteamaculans population. Two of the Serratia proteamaculans-like strains had been chromosomally labeled with GFP to confirm the specifics of their association with the nematode hosts. The associated S. proteamaculans-like isolates isolated from Bath and Chepstow soils were examined further for their pathogenicity to Galleria mellonella and Manduca sexta larvae. Serratia Bath isolates, isolated from Pristionchus were more virulent toward both insect hosts than the Serratia from the Chepstow isolates associated with Steinernema nematodes. This suggests that host specificity may play important role in the virulence of the strain.