Tipping the balance : sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.

Transcriptome analyses of amoebic gill disease-affected atlantic salmon (Salmo salar) tissues reveal localized host gene suppression

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (± SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response. © 2008 Springer Science+Business Media, LLC.

The koala immunological toolkit: Sequence identification and comparison of key markers of the koala (Phascolarctos cinereus) immune response

The koala (Phascolarctos cinereus) is an Australian marsupial that continues to experience significant population declines. Infectious diseases caused by pathogens such as Chlamydia are proposed to have a major role. Very few species-specific immunological reagents are available, severely hindering our ability to respond to the threat of infectious diseases in the koala. In this study, we utilise data from the sequencing of the koala transcriptome to identify key immunological markers of the koala adaptive immune response and cytokines known to be important in the host response to chlamydial infection in other species. This report describes the identification and preliminary sequence analysis of (1) T lymphocyte glycoprotein markers (CD4, CD8); (2) IL-4, a marker for the Th2 response; (3) cytokines such as IL-6, IL-12 and IL-1β, that have been shown to have a role in chlamydial clearance and pathology in other hosts; and (4) the sequences for the koala immunoglobulins, IgA, IgG, IgE and IgM. These sequences will enable the development of a range of immunological reagents for understanding the koala’s innate and adaptive immune responses, while also providing a resource that will enable continued investigations into the origin and evolution of the marsupial immune system.

Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

The role of the multiple banded antigen of ureaplasma parvum in intra-amniotic infection : major virulence factor or decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.

Microbial colonisation of human follicular fluid and adverse in vitro fertilisation outcomes

This study, investigating 263 women undergoing trans-vaginal oocyte retrieval for in vitro fertilisation (IVF) found that microorganisms colonising follicular fluid contributed to adverse IVF (pre-implantation) and pregnancy (post-implantation) outcomes including poor quality embryos, failed pregnancy and early pregnancy loss (< 37 weeks gestation). Some microorganisms also showed in vitro growth patterns in liquid media that appeared to be enhanced by the hormonal stimulation protocol used for oocyte retrieval. Elaborated cytokines within follicular fluid were also associated with adverse IVF outcomes. This study is imperative because infertility affects 16% of the human population and the numbers of couples needing assistance continues to increase. Despite significant improvements in the technical aspects of assisted reproductive technologies (ART), the live birth rate has not increased proportionally. Overt genital tract infection has been associated with both infertility and adverse pregnancy outcomes (including miscarriage and preterm birth) as a direct result of the infection or the host response to it. Importantly, once inflammation had become established, medical treatment often failed to prevent these significant adverse outcomes. Current evaluations of fertility focus on the ovary as a site of steroid hormone production and ovulation. However, infertility as a result of subclinical colonisation of the ovary has not been reported. Furthermore, identification of the microorganisms present in follicular fluid and the local cytokine profile may provide clinicians with an early indication of the prognosis for IVF treatment in infertile couples, thus allowing antimicrobial treatment and/or counselling about possible IVF failure. During an IVF cycle, multiple oocytes undergo maturation in vivo in response to hormonal hyperstimulation. Oocytes for in vitro insemination are collected trans-vaginally. The follicular fluid that bathes the maturing oocyte in vivo, usually is discarded as part of the IVF procedure, but provides a unique opportunity to investigate microbial causes of adverse IVF outcomes. Some previous studies have identified follicular fluid markers that predict IVF pregnancy outcomes. However, there have not been any detailed microbiological studies of follicular fluid. For this current study, paired follicular fluid and vaginal secretion samples were collected from women undergoing IVF cycles to determine whether microorganisms in follicular fluid were associated with adverse IVF outcomes. Microorganisms in follicular fluid were regarded as either "colonisers" or "contaminants"; colonisers, if they were unique to the follicular fluid sample, and contaminants if the same microorganisms were detected in the vaginal and follicular fluid samples indicating that the follicular fluid was merely contaminated during the oocyte retrieval process. Quite unexpectedly, by these criteria, we found that follicular fluid from approximately 30% of all subjects was colonised with bacteria. Fertile and infertile women with colonised follicular fluid had decreased embryo transfer rates and decreased pregnancy rates compared to women with contaminated follicular fluids. The observation that follicular fluid was not always sterile, but contained a diverse range of microorganisms, is novel. Many of the microorganisms we detected in follicular fluid are known opportunistic pathogens that have been detected in upper genital tract infections and are associated with adverse pregnancy outcomes. Bacteria were able to survive for at least 28 weeks in vitro, in cultures of follicular fluid. Within 10 days of establishing these in vitro cultures, several species (Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., Streptococcus spp. and Salmonella entericus) had formed biofilms. Biofilms play a major role in microbial pathogenicity and persistence. The propensity of microbial species to form biofilms in follicular fluid suggests that successful treatment of these infections with antimicrobials may be difficult. Bifidobacterium spp. grew, in liquid media, only if concentrations of oestradiol and progesterone were similar to those achieved in vivo during an IVF cycle. In contrast, the growth of Streptococcus agalactiae and Escherichia coli was inhibited or abolished by the addition of these hormones to culture medium. These data suggest that the likelihood of microorganisms colonising follicular fluid and the species of bacteria involved is influenced by the stage of the menstrual cycle and, in the case of IVF, the nature and dose of steroid hormones administered for the maturation of multiple oocytes in vivo. Our findings indicate that the elevated levels of steroid hormones during an IVF cycle may influence the microbial growth within follicular fluid, suggesting that the treatment itself will impact on the microflora present in the female upper genital tract during pre-conception and early post-conception phases of the cycle. The effect of the host immune response on colonising bacteria and on the outcomes of IVF also was investigated. White blood cells reportedly compose between 5% and 15% of the cell population in follicular fluid. The follicular membrane is semi-permeable and cells are actively recruited as part of the normal menstrual cycle and in response to microorganisms. A previous study investigated follicular fluid cytokines from infertile women and fertile oocyte donors undergoing IVF, and concluded that there were no significant differences in the cytokine concentrations between the two groups. However, other studies have reported differences in the follicular fluid cytokine levels associated with infertile women with endometriosis or polycystic ovary syndrome. In this study, elevated levels of interleukin (IL)-1 á, IL-1 â and vascular endothelial growth factor (VEGF) in vaginal fluid were associated with successful fertilisation, which may be useful marker for successful fertilisation outcomes for women trying to conceive naturally or prior to oocyte retrieval for IVF. Elevated levels of IL-6, IL-12p40, granulocyte colony stimulating factor (GCSF) and interferon-gamma (IFN ã) in follicular fluid were associated with successful embryo transfer. Elevated levels of pro-inflammatory IL-18 and decreased levels of anti-inflammatory IL-10 were identified in follicular fluid from women with idiopathic infertility. Successful fertilisation and implantation is dependent on a controlled pro-inflammatory environment, involving active recruitment of pro-inflammatory mediators to the genital tract as part of the menstrual cycle and early pregnancy. However, ongoing pregnancy requires an enhanced anti-inflammatory environment to ensure that the maternal immune system does not reject the semi-allergenic foetus. The pro-inflammatory skew in the follicular fluid of women with idiopathic infertility, correlates with normal rates of fertilisation, embryo discard and embryo transfer, observed for this cohort, which were similar to the outcomes observed for fertile women. However, their pregnancy rate was reduced compared to fertile women. An altered local immune response in follicular fluid may provide a means of explaining infertility in this cohort, previously defined as 'idiopathic'. This study has found that microorganisms colonising follicular fluid may have contributed to adverse IVF and pregnancy outcomes. Follicular fluid bathes the cumulus oocyte complex during the in vivo maturation process, and microorganisms in the fluid, their metabolic products or the local immune response to these microorganisms may result in damage to the oocytes, degradation of the cumulus or contamination of the IVF culture system. Previous studies that have discounted bacterial contamination of follicular fluid as a cause of adverse IVF outcomes failed to distinguish between bacteria that were introduced into the follicular fluid at the time of trans-vaginal oocyte retrieval and those that colonised the follicular fluid. Those bacteria that had colonised the fluid may have had time to form biofilms and to elicit a local immune response. Failure to draw this distinction has previously prevented consideration of bacterial colonisation of follicular fluid as a cause of adverse IVF outcomes. Several observations arising from this study are of significance to IVF programs. Follicular fluid is not always sterile and colonisation of follicular fluid is a cause of adverse IVF and pregnancy outcomes. Hormonal stimulation associated with IVF may influence whether follicular fluid is colonised and enhance the growth of specific species of bacteria within follicular fluid. Bacteria in follicular fluid may form biofilms and literature has reported that this may influence their susceptibility to antibiotics. Monitoring the levels of selected cytokines within vaginal secretions may inform fertilisation outcomes. This study has identified novel factors contributing to adverse IVF outcomes and that are most likely to affect also natural conception outcomes. Early intervention, possibly using antimicrobial or immunological therapies may reduce the need for ART and improve reproductive health outcomes for all women.

Determining epithelial contribution to in vivo mesenchymal tumour expression signature using species-specific microarray profiling analysis of xenografts

Gene expression profiling using microarrays and xenograft transplants of human cancer cell lines are both popular tools to investigate human cancer. However, the undefined degree of cross hybridization between the mouse and human genomes hinders the use of microarrays to characterize gene expression of both the host and the cancer cell within the xenograft. Since an increasingly recognized aspect of cancer is the host response (or cancer-stroma interaction), we describe here a bioinformatic manipulation of the Affymetrix profiling that allows interrogation of the gene expression of both the mouse host and the human tumour. Evidence of microenvironmental regulation of epithelial mesenchymal transition of the tumour component in vivo is resolved against a background of mesenchymal gene expression. This tool could allow deeper insight to the mechanism of action of anti-cancer drugs, as typically novel drug efficacy is being tested in xenograft systems.

Infection and cellular defense dynamics in a novel 17beta-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (106–107 CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Uropathogenic Escherichia Coli engages CD14-dependent signaling to enable bladder-macrophage-dependent control of acute urinary tract infection

Background CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. Methods We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14−/− mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. Results UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14−/− mice. Exacerbation of infection in Cd14−/− mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. Conclusions This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Cytotoxic Effects and Biocompatibility of Antimicrobial Materials

The rising demand for medical implants for ageing populations and ongoing advancements in medical technology continue to drive the use of implantable devices. Higher implant usage has a consequent increased incidence of implant-related infections, and associated prolonged patient care, pain and loss of limb and other organ function. Numerous antibacterial surfaces have been designed that prevent the onset of biofilm formation, thus reducing or preventing implant-associated infections through inhibiting bacterial adhesion or by killing the organisms that successfully attach to the surface of the implant. Other surfaces have been designed to stimulate a local immune response, promoting the natural clearing of the invading pathogen. The desired antibacterial effects are typically achieved by modulating the surface chemistry and morphology of the implant material, by means of the controlled release of pharmacological agents and bioactive compounds from the surface of the material, or by a combination of both processes. An important issue for any type of antibacterial surface modification lies in balancing the non-fouling, bacteriostatic or bactericidal effects against local and systemic biocompatibility. In this chapter, we will first describe the concept of biocompatibility and its evolution, from devices that do not evoke a negative host response to those that actively drive host regeneration. We will then review the challenges associated with merging the need for an implant material to withstand a bacterial load with those associated with supporting function restoration and tissue healing.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

Host searching behaviour of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae: Opiinae), a polyphagous parasitoid of Dacinae fruit flies (Diptera: Tephritidae)

Diachasmimorpha kraussii (Hymenoptera: Braconidae: Opiinae) is a koinobiont larval parasitoid of dacine fruit flies of the genus Bactrocera (Diptera: Tephritidae) in its native range (Australia, Papua New Guinea, Solomon Islands). The wasp is a potentially important control agent for pest fruit flies, having been considered for both classical and inundative biological control releases. I investigated the host searching, selection and utilisation mechanisms of the wasp against native host flies within its native range (Australia). Such studies are rare in opiine research where the majority of studies, because of the applied nature of the research, have been carried out using host flies and environments which are novel to the wasps. Diachasmimorpha kraussii oviposited equally into maggots of four fruit fly species, all of which coexist with the wasp in its native range (Australia), when tested in a choice trial using a uniform artificial diet media. While eggs laid into Bactrocera tryoni and B. jarvisi developed successfully through to adult wasps, eggs laid into B. cucumis and B. cacuminata were encapsulated. These results suggest that direct larval cues are not an important element in host selection by D. kraussii. Further exploring how D. kraussii locates suitable host larvae, I investigated the role of plant cues in host searching and selection. This was examined in a laboratory choice trial using uninfested fruit or fruit infested with either B. tryoni or B. jarvisi maggots. The results showed a consistent preference ranking among infested fruits by the wasp, with guava and peach most preferred, but with no response to uninfested fruits. Thus, it appears the wasp uses chemical cues emitted in response to fruit fly larval infestation for host location, but does not use cues from uninfested fruits. To further tease apart the role of (i) suitable and non-suitable maggots, (ii) infested and uninfested fruits of different plant species, and (iii) adult flies, in wasp host location and selection, I carried out a series of behavioural tests where I manipulated these attributes in a field cage. These trials confirmed that D. kraussii did not respond to cues in uninfested fruits, that there were consistent preferences by the wasps for different maggot infested fruits, that fruit preference did not vary depending on whether the maggots were physiologically suitable or not suitable for wasp offspring development, and finally, that adult flies appear to play a secondary role as indicators of larval infestation. To investigate wasp behaviour in an unrestrained environment, I concurrently observed diurnal foraging behaviours of both the wasp and one of its host fly in a small nectarine orchard. Wasp behaviour, both spatially and temporally, was not correlated with adult fruit fly behaviour or abundance. This study reinforced the point that infested fruit seems to be the primary cue used by foraging wasps. Wasp and fly feeding and mating was not observed in the orchard, implying these activities are occurring elsewhere. It is highly unlikely that these behaviours were happening within the orchard during the night as both insects are diurnal. As the final component of investigating host location, I carried out a habitat preference study for the wasp at the landscape scale. Using infested sentinel fruits, I tested the parasitism rate of B. tryoni in eucalyptus sclerophyll forest, rainforest and suburbia in South East Queensland. Although, rainforest is the likely endemic habitat of both B. tryoni and D. kraussii, B. tryoni abundance is significantly greater in suburban environments followed by eucalyptus sclerophyll forest. Parasitism rate was found to be higher in suburbia than in the eucalyptus sclerophyll forest, while no parasitism was recorded in the rainforest. This result suggests that wasps orient within the landscape towards areas of high host density and are not restricted by habitat types. Results from the different experiments suggest that host searching, selection and utilisation behaviour of D. kraussii are strongly influenced by cues associated with fruit fly larval feeding. Cues from uninfested fruits, the host larvae themselves, and the adult host flies play minimal roles. The discussion focuses on the fit of D. kraussii to Vinson’s classical parasitoid host location model and the implications of results for biological control, including recommendations for host and plant preference screening protocols and release regimes.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.