Ageing reduces fitness, but how ageing evolves is still unclear. Evolutionary theory of ageing hinges on the fundamental principal that the force of natural selection declines with age. This principle has yielded two important predictions: 1) the evolution of faster ageing in populations under high rate of extrinsic mortality; and 2) the evolution of faster ageing in a sex that experiences higher rates of extrinsic mortality. However, an emerging new theory argues that when the extrinsic mortality is not random but instead selects on traits showing positive genetic correlation with lifespan, increased mortality should lead to the evolution of increased lifespan. Such condition-dependent mortality is also expected to increase the robustness in the population, resulting in increased deceleration of mortality in late-life. Similarly, high sex-specific mortality can result in increased sex-specific selection on traits that have positive pleiotropic effects on lifespan in the affected sex. This thesis is based on two experimental evolution studies in Caenorhabditis remanei. The first experiment was designed to disentangle the effects of the rate (high or low) and the source (random or condition-dependent) of mortality on the evolution of lifespan and ageing. Reduced lifespan evolved under high rate of random mortality, whereas high condition-dependent mortality, imposed by heat-shock, led to the evolution of increased lifespan (Paper I). However, while female reproduction increased under condition-dependent mortality, male reproduction suffered, suggesting a role for sexual antagonism in maintaining genetic variation for fitness (Paper II). Besides, long lifespan and high fecundity evolved at a cost of slow juvenile growth rate in females (Paper III). Moreover, high condition-dependent mortality led to the evolution of lower rate of intrinsic mortality in late-life (Paper IV). The second experiment showed that evolution of sexual dimorphism in lifespan is driven by the factors that cause sex-specific mortality and cannot be predicted from differences in mortality rate alone. Specifically, high condition-dependent mortality renders males less prone to ageing than females despite higher rates of male mortality (Paper V). The strength of this thesis is the reconfirmation of the earlier findings combined with support for the new theory. Rather than further complicating the matter, the inclusion of the new ideas should help explain some empirical results that are inconsistent with the classic theory, as well as provide a more comprehensive picture of ageing evolution.

The combined survival status of the insured lives is a critical problem when pricing and reserving insurance products with more than one life. Our preliminary experience examination of bivariate annuity data from a large Canadian insurance company shows that the relative risk of mortality for an individual increases after the loss of his/her spouse, and that the increase is especially dramatic shortly after bereavement. This preliminary result is supported by the empirical studies over the past 50 years, which suggest dependence between a husband and wife. The dependence between a married couple may be significant in risk management of joint-life policies. This dissertation progressively explores Markovian models in pricing and risk management of joint-life policies, illuminating their advantages in dependent modeling of joint time-until-death (or other exit time) random variables. This dissertation argues that in the dependent modeling of joint-life dependence, Markovian models are flexible, transparent, and easily extended. Multiple state models have been widely used in historic data analysis, particularly in the modeling of failures that have event-related dependence. This dissertation introduces a ¡°common shock¡± factor into a standard Markov joint-life mortality model, and then extends it to a semi-Markov model to capture the decaying effect of the "broken heart" factor. The proposed models transparently and intuitively measure the extent of three types of dependence: the instantaneous dependence, the short-term impact of bereavement, and the long-term association between lifetimes. Some copula-based dependence measures, such as upper tail dependence, can also be derived from Markovian approaches. Very often, death is not the only mode of decrement. Entry into long-term care and voluntary prepayment, for instance, can affect reverse mortgage terminations. The semi-Markov joint-life model is extended to incorporate more exit modes, to model joint-life reverse mortgage termination speed. The event-triggered dependence between a husband and wife is modeled. For example, one spouse's death increases the survivor's inclination to move close to kin. We apply the proposed model specifically to develop the valuation formulas for roll-up mortgages in the UK and Home Equity Conversion Mortgages in the US. We test the significance of each termination mode and then use the model to investigate the mortgage insurance premiums levied on Home Equity Conversion Mortgage borrowers. Finally, this thesis extends the semi-Markov joint-life mortality model to having stochastic transition intensities, for modeling joint-life longevity risk in last-survivor annuities. We propose a natural extension of Gompertz' law to have correlated stochastic dynamics for its two parameters, and incorporate it into the semi-Markov joint-life mortality model. Based on this preliminary joint-life longevity model, we examine the impact of mortality improvement on the cost of a last survivor annuity, and investigate the market prices of longevity risk in last survivor annuities using risk-neutral pricing theory.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Wolbachia is a maternally inherited α-proteobacteria that lives an obligate intracellular lifestyle in its eukaryotic hosts. Wolbachia pipientis was first identified in the 1920’s in the mosquito Culex pipiens; Wolbachia has since been detected in a broad range of hosts. Wolbachia induces a number of diverse reproductive manipulations in many of its hosts, implicating Wolbachia in control strategies for target species. However, changes in host bacterial interactions can evolve toward mutualism, and this requires investigation in target systems. / This first part of this work examines changes in the Drosophila simulans-Wolbachia Riverside system in California. This system, studied for a period of more than two decades, provides a rare example in nature of a symbiotic relationship shifting from parasitism to mutualism. In my study, no changes in overall estimates of infection frequency, maternal transmission or cytoplasmic incompatibility were found, however there appears to be heterogeneity in maternal transmission rates and the ability of males to induce cytoplasmic incompatibility (CI). The source of CI variation in one isofemale line was subsequently investigated through reciprocal backcrossing with an old Wolbachia-infected strain, Riv88, and the variation was attributed to the Wolbachia genome. Further investigations are required to determine the extent of CI variation in the wild. These findings significant implications for the many applied projects that intend to exploit Wolbachia for its ability to manipulate host reproductive systems. / The second part of this work investigates the extent to which evolution of a host-bacterial symbiosis can influence the effectiveness of control strategies using Wolbachia. A particular strain of Wolbachia, named popcorn (wMelPop), has been isolated for its ability to reduce host longevity. This bacterium has the capacity to alter the age structure of the target host populations. As many vector-borne diseases (eg: Dengue Fever, Malaria) require an incubation period within the vector prior to transmission to the final host, a reduction in the lifespan of vector populations has the potential to lower transmission levels. The long term stability of this bacterial strain has been investigated in its native host (Drosophila melanogaster), and a novel host (D. simulans). The longevity-shortening phenotype and several life-history traits that might influence the effectiveness of control strategies have been examined. Host genetic background and the indirect selection on the longevity phenotype (via early or late fecundity selection) were found to influence multiple life-history traits in these species. The source of this variation in D. melanogaster is attributed primarily to the host, but can also be influenced by Wolbachia. Strong host background effects were noted in D. simulans, which highlights the importance of testing multiple backgrounds for suitability of use in applied projects. / This work has allowed for greater understanding of the potential for evolution within host-symbiont systems, and can be used to provide a framework for undertaking projects that will use Wolbachia as biological control agents. Future projects should consider the potential for evolution in detail under laboratory conditions before control strategies are implemented in the wild.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Wolbachia is a maternally inherited α-proteobacteria that lives an obligate intracellular lifestyle in its eukaryotic hosts. Wolbachia pipientis was first identified in the 1920’s in the mosquito Culex pipiens; Wolbachia has since been detected in a broad range of hosts. Wolbachia induces a number of diverse reproductive manipulations in many of its hosts, implicating Wolbachia in control strategies for target species. However, changes in host bacterial interactions can evolve toward mutualism, and this requires investigation in target systems. / This first part of this work examines changes in the Drosophila simulans-Wolbachia Riverside system in California. This system, studied for a period of more than two decades, provides a rare example in nature of a symbiotic relationship shifting from parasitism to mutualism. In my study, no changes in overall estimates of infection frequency, maternal transmission or cytoplasmic incompatibility were found, however there appears to be heterogeneity in maternal transmission rates and the ability of males to induce cytoplasmic incompatibility (CI). The source of CI variation in one isofemale line was subsequently investigated through reciprocal backcrossing with an old Wolbachia-infected strain, Riv88, and the variation was attributed to the Wolbachia genome. Further investigations are required to determine the extent of CI variation in the wild. These findings significant implications for the many applied projects that intend to exploit Wolbachia for its ability to manipulate host reproductive systems. / The second part of this work investigates the extent to which evolution of a host-bacterial symbiosis can influence the effectiveness of control strategies using Wolbachia. A particular strain of Wolbachia, named popcorn (wMelPop), has been isolated for its ability to reduce host longevity. This bacterium has the capacity to alter the age structure of the target host populations. As many vector-borne diseases (eg: Dengue Fever, Malaria) require an incubation period within the vector prior to transmission to the final host, a reduction in the lifespan of vector populations has the potential to lower transmission levels. The long term stability of this bacterial strain has been investigated in its native host (Drosophila melanogaster), and a novel host (D. simulans). The longevity-shortening phenotype and several life-history traits that might influence the effectiveness of control strategies have been examined. Host genetic background and the indirect selection on the longevity phenotype (via early or late fecundity selection) were found to influence multiple life-history traits in these species. The source of this variation in D. melanogaster is attributed primarily to the host, but can also be influenced by Wolbachia. Strong host background effects were noted in D. simulans, which highlights the importance of testing multiple backgrounds for suitability of use in applied projects. / This work has allowed for greater understanding of the potential for evolution within host-symbiont systems, and can be used to provide a framework for undertaking projects that will use Wolbachia as biological control agents. Future projects should consider the potential for evolution in detail under laboratory conditions before control strategies are implemented in the wild.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 79-91). Also available on the Internet.