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ItemThe genetic and morphological characterisation of the zebrafish intestinal mutant setebos(University of Melbourne, 2010)Colorectal cancer (CRC) arises by mutation of tumour suppressors and oncogenes that deregulate the genetic programmes that control intestinal turnover and homeostasis. The zebrafish intestinal epithelium shares extensive phenotypic and genetic homology with the mammalian intestine, providing a genetically tractable model organism for the discovery and characterisation of novel genes required for intestinal development. Increased knowledge of the genetic control of intestinal development may reveal new therapeutic avenues for the treatment of CRC. The Liverplus mutagenesis screen identified zebrafish embryos defective in endoderm organ morphogenesis, including the intestinal mutant setebos, described herein. This study reports the positional cloning of the setebos mutant locus, identifying a premature stop codon as the responsible mutation in the gene nucleolar protein 8 (nol8). Microinjection of wildtype nol8 mRNA rescued setebos developmental abnormalities whereas morpholino oligonucleotide-mediated knockdown of Nol8 phenocopied setebos morphology in wildtype embryos, providing functional proof that nol8 is the mutated locus underlying the setebos phenotype. This study demonstrated that Nol8 is essential for zebrafish survival and that nol8 mRNA is widely expressed in actively proliferating tissues of the zebrafish embryo at 72 hours post fertilisation (hpf), when developmental abnormalities in setebos mutants first become apparent. Northern blot analyses revealed that setebos mutants accumulate an intermediate ribosomal RNA (rRNA) orthologous to mammalian 36S at the expense of downstream 32S rRNA; defects predicted to result in reduced biogenesis of 28S rRNA. Bioanalyzer analyses confirm that setebos mutants are preferentially impaired for 28S biogenesis. 28S, 5.8S and 18S rRNA molecules combine with ribosomal proteins to form functional ribosomes, which are essential for translation and cellular growth. Defects in ribosome biogenesis have been shown to activate the tumour suppressor Tp53, resulting in cell cycle arrest. Tp53 is activated in the setebos mutant, resulting in upregulation of pro-cell cycle arrest genes, including the N-terminally truncated isoform of Tp53, ?113tp53. While this provides a mechanism for the hypoplasia observed in the tissues of the setebos embryo, including the intestinal epithelium, craniofacial cartilages, eyes, liver and pancreas, inactivation of Tp53 signalling in the setebos mutant failed to obviously alter setebos morphology. Nevertheless, activation of Tp53 signalling was found to significantly extend survival of setebos embryos, identifying an important biological role for Tp53 in cells undergoing ribosomal stress. Multiple human syndromes have been linked to abnormal ribosome biogenesis (ribosomopathies), and several predispose to cancer. Zebrafish haploinsufficient for 17 different ribosomal proteins also display increased cancer susceptibility (Amsterdam et al. 2004; Lai et al. 2009). Tp53 is inactivated in these zebrafish tumours, suggesting that inactivation of Tp53 may be a key event in tumour formation in these animals. In summary, setebos provides an ideal in vivo model to further characterise the link between defective ribosome biogenesis, Tp53 signalling and cancer. Since disruption of Nol8 function preferentially impairs the growth of several highly proliferative embryonic zebrafish tissues in the absence of Tp53 signalling, inhibition of Nol8 may provide a therapeutic avenue for the treatment of Tp53 mutant tumours.
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ItemStructure(University of Melbourne, 2010)MLK2 is a serine/threonine kinase with a N-terminal SH3 domain and a centrally located dual leucine zipper region that defines the mixed lineage kinase family. Motifs for interaction with the small Rho-like GTPases, 14-3-3 signalling adaptor proteins, as well as for nuclear import and export have been identified in MLK2 and other MLK family members, providing the capacity for a large number of diverse interactions. MLK2 extracted from over-expressing cells is catalytically active and long-term over-expression is lethal. A study of the contribution of the SH3 domain, catalytic domain and dual leucine zipper region to the overall activity of MLK2 was undertaken. This investigation utilised targeted mutagenesis to alter the coding sequence of conserved amino acids to produce non-functional and constitutively active protein domains. The SH3 domain contributes to the regulation of catalytic activity by binding within the protein C-terminal domain. Once released from auto-inhibition, the SH3 domain interacts with dynamin and is capable of activating dynamin I GTPase activity, thereby increasing the rates of dynamin mediated endocytosis. Both auto-inhibition and dynamin activation is ablated by substitution of a praline residue within the conserved binding cleft of the SH3 domain. Dim�risation via the dual leucine zipper region is required to achieve full catalytic activity. Substitution within the catalytic domain produced a kinase-inactive protein that was used throughout the study. In addition, this inactive variant also demonstrated the requirement for phosphorylation of the activation loop prior to an increase in catalytic activity. Membrane localisation of MLK2 was demonstrated to be independent of catalytic activity and SH3 domain or leucine zipper binding capacity. MLK2 is a member of the MAPKKK family capable of activating the c-jun N terminal kinases via MKK4 and MKK7. The use of kinase negative MKK4 confirmed the ability of MLK2 to signal to JNK through an alternative MKK protein. Kinase negative MLK2 was a target for JNK phosphorylation producing a pattern of phosphopeptide TLC-TLE spots with commonalities to autophosphorylated MLK2. Phosphorylation was concentrated within the C-terminal half of the protein where no large functional domains have been identified but a number of smaller motifs are found. Retrophosphorylation was proposed to have a positive effect on MLK2, sustaining activity and signalling that ultimately leads to apoptosis. Stable expression of GFP tagged MLK2 was only achieved with kinase inactive or truncated protein coding constructs, confirming the lethal effect of over-expressing active MLK2. Kinase negative MLK2 expression decreased cell doubling rates, increased the number of fine membrane projections and trafficked to the nucleus in a cell-cycle dependent manner. The CRIB domain allowed for interaction between Cdc42, Rad and MLK2. MLK2 was proposed to influence cytoskeletal remodelling in response to Cdc42 and Rad activation leading to stimulation of the JNK pathway. Finally, MLK2 was has been shown to have a role in the progression of the neurodegenerative pathology of Alzheimer�s disease. Stimulation of the JNK pathway, coupled with the ability to mediate receptor activation by modulation of rates of endocytosis theoretically places MLK2 in a key position in the response to excitory amino acid excitotoxicity, in Huntington�s disease, and to apoER2 activation in Alzheimer s disease. The characterisation of the contributions made by SH3 domain, catalytic domain and dual leucine zipper region to the overall activity of MLK2 will allow for the design and optimisation of novel therapeutic targets aimed at reducing MLK2 activity, opening up new scope for disease management.
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ItemThe roles of a novel group of plastid transporters in glutathione metabolism in arabidopsis(University of Melbourne, 2010)The tripeptide glutathione (GSH) is an important compound with a broad spectrum of functions in plants, including stress responses and is a key element of the intracellular redox buffer that protects cells from oxidation. In Arabidopsis GSH is synthesized in two enzymatic steps by GSH1, found exclusively in the plastids, forming the pathway intermediate ?-glutamylcysteine (?-EC) and then by GSH2 which is located in both plastids and cytosol. This suggests a mechanism for ?-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is believed that the cytosol may be the main compartment for GSH biosynthesis. In addition, GSH may be efficiently transported between the two compartments to maintain proper homeostasis. However, comparatively little is understood about the mechanisms that control and interconnect these two subcellular GSH pools. The identification and characterization of a novel family of transporters, the CRT Like Transporters (CLTs), has provided further insights into glutathione metabolism in Arabidopsis. The CLT1 gene was originally identified in a screen for mutants resistant to L-Buthionine-SR-sulfoximine (BSO), a synthetic compound used to specifically inhibit GSH1 and GSH biosynthesis. Two paralogues in Arabidopsis, CLT2 and CLT3, were identified and all three CLTs are localized to the plastids. Previous studies have shown the clt1clt2clt3 triple mutant is GSH-deficient in roots and cadmium-sensitive suggesting a role for the CLTs in transporting ?-EC and/or GSH between the plastid and cytosol compartments. In this study redirecting GSH1 activity or GSH biosynthesis exclusively to the cytosol by complementing the Arabidopsis gsh1 and gsh2 mutants with E. coli GSHA and GSHB, respectively, has no significant impact on phenotypes or stress resistance suggesting efficient exchange of ?-EC and GSH between the plastid and cytosol compartments. The use of redox-sensitive roGFP2 reporters in combination with confocal imaging with monochlorobimane and measurements of cytosolic thiols has demonstrated that the clt1clt2clt3 mutant is GSH-deficient in the cytosol but not the plastid in both roots and shoots consistent with a predicted role for CLTs in ?-EC and/or GSH transport. The Arabidopsis gsh2 mutant accumulates high levels of ?-EC in the cytosol. However, the combination of the clt1clt2clt3 mutations with gsh2 did not decrease ?-EC levels suggesting CLTs may not contribute to ?-EC transport in vivo. To further study the roles of the CLTs separately on ?-EC and GSH transport in planta different clt mutations were introduced into transgenic lines expressing plastid- or cytosol-targeted GSH2. These observations suggest that de novo GSH biosynthesis in wildtype Arabidopsis occurs largely in the plastids and that CLT2 and CLT1/CLT3 are mainly involved in ?-EC and GSH export, respectively, in planta.
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ItemInvestigating the mechanisms of zinc homeostasis in Arabidopsis thaliana(University of Melbourne, 2010)Zn is an element essential for all forms of life, and either Zn-deficiency or excess Zn can have serious biological consequences. In plants, Zn levels are controlled by a number of physiological and cellular mechanisms to maintain appropriate physiological concentrations of Zn. This study further characterises the HMA2 and HMA4 transporters by developing a novel technique using the Zn fluorophore Zinpyr-1. A procedure for the use of Zinpyr-1 in Arabidopsis roots was developed and validated which allowed the visualisation and relative quantification of Zn. This technique showed that both hma4 and hma2/hma4 mutants differed in the amount of Zn and its distribution compared to wildtype or hma2. This correlates to previous data showing that hma4 and hma2/hma4 are Zn-deficient in shoots but accumulate Zn in roots. Thus we can say that this Zn- deficiency in shoot occurs due to an inability to load Zn into the xylem for transport to the shoot. To investigate the regulation of Zn-homeostasis, Zn-deficient and hma2/hma4 plants were compared to wildtype in micro-array experiments to test whether plants respond systemically to Zn- deficiency. hma2/hma4 plants are Zn-deficient in shoots but over-accumulate Zn in roots, so genes co-regulated in hma2/hma4 roots and shoots, and genes co-regulated in hma2/hma4 roots and Zn- deficient wildtype roots were identified. 52 and 27 genes were identified in these comparisons respectively as regulated >2-fold in the same direction. These were considered robust as their co-regulation was observed in four measurements, two technical replicates of two biological replicates. promoter
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ItemPopulation genetics and ecology of the lucerne flea, sminthurus viridis, with applications for control(University of Melbourne, 2010)The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae), is a major pest of broadacre grains and pasture throughout southern Australia. Current control methods for this pest rely heavily on the prophylactic use of broad spectrum pesticides and are unlikely to be sustainable. Development of an integrated pest management (IPM) strategy is needed. However, this requires a greater understanding of the biology, ecology and genetics of S. viridis. This thesis aims to address this by examining the population genetics and life-cycle of this pest, as well as investigating chemical and biological control methods. Laboratory pesticide bioassays were used to compare the response of S. viridis with the redlegged earth mite (Halotydeus destructor) to six broad spectrum pesticides. Sminthurus viridis displayed significantly higher pesticides tolerance than H. destructor and the tolerance differences were greater for the synthetic pyrethroids than the organophosphorous pesticides. This indicates that application rates for S. viridis should be greater than H. destructor and that organophosphates are likely to provide the most effective control of this pest. Seasonal abundance patterns and summer diapause response of S. viridis in south-eastern Australia were investigated using field and shade-house experiments. The seasonal activity agreed with previous studies and parameters predicting autumn emergence were defined. A summer-diapausing egg stage was also confirmed, with diapause eggs accumulating across the season. These results suggest autumn emergence of S. viridis can be predicted for target control strategies, but late-season spraying strategies may be difficult to implement. The species status, mode of reproduction and population genetics of S. viridis were examined using microsatellite markers I developed, together with allozymes and a mitochondrial DNA marker. A single, sexually reproducing species was found with limited gene flow between Australian populations. Genetic similarity between some distant populations was also shown, indicating that wind or human-mediated long-distance dispersal occurs. These findings suggest that management strategies may be more effective when locally developed and need to consider the effects of human- mediated dispersal. The impact of the predatory mite, Bdellodes lapidaria, for controlling S. viridis and its susceptibility to several pesticides were assessed using laboratory and field experiments. Bdellodes lapidaria was more susceptible to the synthetic pyrethroids in the laboratory, but was not affected by pesticide applications in the field. In addition, no detectable predator-prey relationship was found between these species. These results indicate that B. lapidaria is not strongly susceptible to pesticides in the field and may not be an effective predator of S. viridis. Overall, this thesis contributes important knowledge of the pesticide tolerance, life- cycle, population genetics and biological control of S. viridis. These findings highlight ways to improve the effectiveness and sustainability of control methods and bring closer the development of an integrated pest management strategy for S. viridis.
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ItemPopulation genetics and ecology of the lucerne flea, Sminthurus viridis, with applications for control( 2010)The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae), is a major pest of broadacre grains and pasture throughout southern Australia. Current control methods for this pest rely heavily on the prophylactic use of broad spectrum pesticides and are unlikely to be sustainable. Development of an integrated pest management (IPM) strategy is needed. However, this requires a greater understanding of the biology, ecology and genetics of S. viridis. This thesis aims to address this by examining the population genetics and life-cycle of this pest, as well as investigating chemical and biological control methods. Laboratory pesticide bioassays were used to compare the response of S. viridis with the redlegged earth mite (Halotydeus destructor) to six broad spectrum pesticides. Sminthurus viridis displayed significantly higher pesticides tolerance than H. destructor and the tolerance differences were greater for the synthetic pyrethroids than the organophosphorous pesticides. This indicates that application rates for S. viridis should be greater than H. destructor and that organophosphates are likely to provide the most effective control of this pest. Seasonal abundance patterns and summer diapause response of S. viridis in southeastern Australia were investigated using field and shade-house experiments. The seasonal activity agreed with previous studies and parameters predicting autumn emergence were defined. A summer-diapausing egg stage was also confirmed, with diapause eggs accumulating across the season. These results suggest autumn emergence of S. viridis can be predicted for target control strategies, but late-season spraying strategies may be difficult to implement. The species status, mode of reproduction and population genetics of S. viridis were examined using microsatellite markers I developed, together with allozymes and a mitochondrial DNA marker. A single, sexually reproducing species was found with limited gene flow between Australian populations. Genetic similarity between some distant populations was also shown, indicating that wind or human-mediated longdistance dispersal occurs. These findings suggest that management strategies may be more effective when locally developed and need to consider the effects of humanmediated dispersal. The impact of the predatory mite, Bdellodes lapidaria, for controlling S. viridis and its susceptibility to several pesticides were assessed using laboratory and field experiments. Bdellodes lapidaria was more susceptible to the synthetic pyrethroids in the laboratory, but was not affected by pesticide applications in the field. In addition, no detectable predator-prey relationship was found between these species. These results indicate that B. lapidaria is not strongly susceptible to pesticides in the field and may not be an effective predator of S. viridis. Overall, this thesis contributes important knowledge of the pesticide tolerance, lifecycle, population genetics and biological control of S. viridis. These findings highlight ways to improve the effectiveness and sustainability of control methods and bring closer the development of an integrated pest management strategy for S. viridis.
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ItemIdentification and characterisation of small molecule inhibitors of PRMT5( 2010)Reactivation of fetal (γ−) globin gene expression is an attractive therapeutic option for various β-globin hemoglobinopathies. Symmetric methylation of H4R3 catalysed by the PRMT5 enzyme has been implicated in repressing γ−globin gene expression. Based on this, we have pursued a small molecule inhibitor of PRMT5 for reactivation of γ−globin. A radiometric assay was developed and miniaturized to screen a 100,000 chemical compound library. A second screen employing analogues of the methyl donor, S-adenosyl- methionine (SAM) was also performed. Active compounds were then analysed to determine their potential to upregulate expression of γ−globin in human erythroid progenitor cells. The work performed establishes the platform for the development of novel therapeutics for the treatment of the various β-hemoglobinopathies.
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ItemIdentification and characterisation of copper homeostasis genes( 2010)Copper is essential for life, yet also potentially toxic in excess. Copper homeostasis is therefore regulated at the cellular, tissue and organismal levels. Studies with eukaryotic model systems, primarily yeast and mammals, have identified conserved mechanisms for copper uptake, distribution, sequestration and efflux. Nevertheless, there is much we do not know about how copper levels are sensed and regulated. Additional proteins involved in copper homeostasis under both ‘normal’ and ‘diseased’ conditions remain to be identified. The emergence of Drosophila melanogaster as a bona fide model system for the study of copper homeostasis has coincided with research aiming to identify and characterise novel copper regulatory genes in this organism. Whereas others have focused on in vivo studies, the studies reported in this thesis are focussed on cultured D. melanogaster S2 cells. The first aim of this project was to use cDNA microarrays to identify genes transcriptionally regulated by copper levels in S2 cells. The second and third aims were to characterise the function and localisation of novel copper homeostasis genes in vitro and in vivo respectively. Initial characterisation of D. melanogaster S2 cells found these cells express orthologues of key mammalian copper regulatory genes. Copper uptake primarily occurs via Ctr1A and Ctr1B, the orthologues of human Ctr1. Copper efflux occurs via DmATP7, the sole D. melanogaster orthologue of the mammalian P-type ATPases, ATP7A and ATP7B. S2 cells are highly copper tolerant and primarily rely on metallothionein-mediated copper sequestration and copper efflux to maintain homeostasis. Whereas ATP7A and ATP7B undergo copper-induced trafficking between the trans-Golgi network and plasma membrane of mammalian cells, this does not appear to occur with endogenous DmATP7 in cultured D. melanogaster cells. Interestingly, when expressed in mammalian cells DmATP7 does undergo copper-induced trafficking to the plasma membrane and can facilitate copper efflux, demonstrating functional conservation of localisation and trafficking motifs in these P-type ATPases. Malvolio, the orthologue of Divalent Metal ion Transporter 1, also contributes to copper uptake in S2 cells and D. melanogaster, with impaired function associated with sensitivity to copper limitation. D. melanogaster therefore utilises this general metal transporter in addition to the copper-specific Ctr1 pathway. cDNA microarrays were used to identify genes transcriptionally regulated by copper in S2 cells, with RNA interference used to determine whether candidate genes could affect copper homeostasis. Several components of the COPI vesicle trafficking pathway, including ADP-ribosylation factor 1 (Arf1), were found to affect copper levels in S2 cells. Arf1 was found to have a conserved role in regulating copper uptake in cultured mammalian and D. melanogaster cells, and this is likely to be mediated via the localisation of Ctr1 at the plasma membrane. Taken together these studies demonstrate the value of D. melanogaster S2 cells for the study of copper homeostasis, thereby complementing the D. melanogaster model system. These novel findings should stimulate additional research in both D. melanogaster and mammalian systems and facilitate a greater understanding of copper homeostasis.
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ItemThe biology, ecology and population genetics of Balaustium medicagoense and Bryobia spp.; emerging mite pests of grains in southern Australia( 2010)Balaustium medicagoense (Acari: Erythraeidae) and Bryobia spp. (Acari: Tetranychidae) have recently been identified as emerging pests of winter crops and pastures by the Australian grains industry. The relative importance of these mites and damage caused by them in Australia has markedly increased in the last decade, and pest reports indicate extensive damage to a variety of winter grain crops and pastures. Furthermore, pest reports suggest that these mites have been difficult to control in the field, with some pesticides described as being ineffective. Despite this, there is very little information about the biology and control of these species, making it difficult to devise effective and sustainable management strategies. This thesis addresses some of these concerns by investigating various aspects of the biology, ecology and genetics of these mites in broad acre agriculture in southern Australia. Laboratory bioassays were undertaken to examine the response of Ba. medicagoense and B. sp. I (along with the redlegged earth mite (Halotydeus destructor) as a point of reference) to several currently registered pesticides against earth mites. Balaustium medicagoense and B. sp. I generally had a much greater level of tolerance to the pesticides tested than H. destructor. These mites therefore have a high natural tolerance to currently registered pesticides and may prove difficult to control in the field. These findings suggest other strategies that are not reliant on chemicals should be considered for control of Ba. medicagoense and B. sp. I. The effects of different crop plants on the survival and reproduction of Ba. medicagoense and Bryobia spp. (B. sp. VIII and B. sp. IX) and the plant damage caused by these mites were investigated under shade-house conditions. In addition information from field surveys and pest report bulletins was collated to test for outbreak patterns. The findings showed that these mites were polyphagous, attacking a variety of agriculturally important plants as well as numerous weeds within southern Australia. Therefore, these mites have the potential to be important pests on several winter grain crops and pasture, but there are important differences in host responses that might assist in management strategies such as targeted crop rotations and weed management. The distribution, life cycle, seasonal abundance patterns and the pest status of Ba. medicagoense and Bryobia spp. were examined in southern Australia. Both mites are widely distributed throughout Mediterranean-type climate areas within southern Australia. Balaustium medicagoense has two generations per year and was active from March until December, with a likely diapause period in summer. There were differences in the seasonal abundance and life cycle of two species of Bryobia (B. sp. IX and B. sp. I). Bryobia sp. IX had two generations per year, was active from March until December, and was likely to be in diapause over the summer months. Bryobia sp. I did not have a diapause stage, was active year round and had approximately four generations per year. Seasonal abundance patterns of Ba. medicagoense and B. sp. IX overlapped with those of the major pest mite species H. destructor and Penthaleus major. A survey of pest outbreaks and chemical control failures showed that while H. destructor and the Penthaleus species remained important pests, outbreaks and control failures involving Ba. medicagoense and Bryobia spp. had increased over the last decade. The species/strain status of Balaustium and Bryobia mites from broad acre environments was examined in southern Australia using a combination of mitochondrial and nuclear DNA sequence data. In addition, the reproductive mode and genetic diversity of Ba. medicagoense was examined using the amplified fragment length polymorphism (AFLP) method. Findings showed that Ba. medicagoense is the only species present in grain crops, pastures and roadsides within southern Australia, and at least seven species of Bryobia mites are present. The AFLP data revealed that Ba. medicagoense reproduces asexually and that genetic diversity was low with only 10 genotypes found from 16 populations. Overall, the data presented in this thesis helps to explain why Ba. medicagoense and Bryobia spp. are emerging pests and points to ways in which these mites may be controlled in the future. The results show that these mites are difficult to control with currently registered pesticides, have a widespread distribution, can potentially increase in abundance rapidly under favourable conditions and persist across a wider range of seasonal conditions than other major pest earth mites. However, there are differences in pesticide susceptibilities and plant host responses that have the potential to be exploited when developing targeted control options for these pest mites.
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ItemEvolution of Wolbachia-Drosophila interactions and implications for Wolbachia-based biocontrol( 2010)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.