Local introduction and heterogeneous spatial spread of dengue-suppressing Wolbachia through an urban population of Aedes aegypti.

Dengue-suppressing Wolbachia strains are promising tools for arbovirus control, particularly as they have the potential to self-spread following local introductions. To test this, we followed the frequency of the transinfected Wolbachia strain wMel through Ae. aegypti in Cairns, Australia, following releases at 3 nonisolated locations within the city in early 2013. Spatial spread was analysed graphically using interpolation and by fitting a statistical model describing the position and width of the wave. For the larger 2 of the 3 releases (covering 0.97 km2 and 0.52 km2), we observed slow but steady spatial spread, at about 100-200 m per year, roughly consistent with theoretical predictions. In contrast, the smallest release (0.11 km2) produced erratic temporal and spatial dynamics, with little evidence of spread after 2 years. This is consistent with the prediction concerning fitness-decreasing Wolbachia transinfections that a minimum release area is needed to achieve stable local establishment and spread in continuous habitats. Our graphical and likelihood analyses produced broadly consistent estimates of wave speed and wave width. Spread at all sites was spatially heterogeneous, suggesting that environmental heterogeneity will affect large-scale Wolbachia transformations of urban mosquito populations. The persistence and spread of Wolbachia in release areas meeting minimum area requirements indicates the promise of successful large-scale population transformation.

Field evaluation of the establishment potential of wMelPop Wolbachia in Australia and Vietnam for dengue control.

BACKGROUND: Introduced Wolbachia bacteria can influence the susceptibility of Aedes aegypti mosquitoes to arboviral infections as well as having detrimental effects on host fitness. Previous field trials demonstrated that the wMel strain of Wolbachia effectively and durably invades Ae. aegypti populations. Here we report on trials of a second strain, wMelPop-PGYP Wolbachia, in field sites in northern Australia (Machans Beach and Babinda) and central Vietnam (Tri Nguyen, Hon Mieu Island), each with contrasting natural Ae. aegypti densities. METHODS: Mosquitoes were released at the adult or pupal stages for different lengths of time at the sites depending on changes in Wolbachia frequency as assessed through PCR assays of material collected through Biogents-Sentinel (BG-S) traps and ovitraps. Adult numbers were also monitored through BG-S traps. Changes in Wolbachia frequency were compared across hamlets or house blocks. RESULTS: Releases of adult wMelPop-Ae. aegypti resulted in the transient invasion of wMelPop in all three field sites. Invasion at the Australian sites was heterogeneous, reflecting a slower rate of invasion in locations where background mosquito numbers were high. In contrast, invasion across Tri Nguyen was relatively uniform. After cessation of releases, the frequency of wMelPop declined in all sites, most rapidly in Babinda and Tri Nguyen. Within Machans Beach the rate of decrease varied among areas, and wMelPop was detected for several months in an area with a relatively low mosquito density. CONCLUSIONS: These findings highlight challenges associated with releasing Wolbachia-Ae. aegypti combinations with low fitness, albeit strong virus interference properties, as a means of sustainable control of dengue virus transmission.

Wolbachia pipientis: an expanding bag of tricks to explore for disease control.

Wolbachia pipientis are maternally inherited, endosymbiotic bacteria that are widespread among insects. Two recent studies have demonstrated that Wolbachia inhibits the ability of medically significant pathogens, including filarial nematodes, dengue virus and Plasmodium to form infections in the mosquito vector, Aedes aegypti. We highlight the added value of these traits for Wolbachia based biocontrol strategies and evaluate the evidence for the idea that the insect immune response is responsible for the pathogen inhibition.

Field validation of a transcriptional assay for the prediction of age of uncaged Aedes aegypti mosquitoes in Northern Australia.

BACKGROUND: New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. METHODOLOGY/PRINCIPAL FINDINGS: We produced "free-range" test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R(2) value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. CONCLUSIONS/SIGNIFICANCE: The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito survivorship or lifespan reduction are crucial to their success. The approximate cost of the method was US$7.50 per mosquito and 60 mosquitoes could be processed in 3 days. The assay is based on conserved genes and modified versions are likely to support similar investigations of several important mosquito and other disease vectors.

Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes.

Wolbachia strain wMelPop reduces the longevity of its Drosophila melanogaster host and, when introduced into the mosquito Aedes aegypti, halves its life span. We show that wMelPop induces up-regulation of the mosquito's innate immune system and that its presence inhibits the development of filarial nematodes in the mosquito. These data suggest that wMelPop could be used in the global effort to eliminate lymphatic filariasis and possibly for the control of other mosquito-borne parasites where immune preactivation inhibits their development. The cost of constitutive immune up-regulation may contribute to the life-shortening phenotype.

Short-term starvation of immune deficient Drosophila improves survival to gram-negative bacterial infections.

BACKGROUND: Primary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-kappaB) pathway. Since the TIR-NF-kappaB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions. METHODOLOGY/PRINCIPAL FINDINGS: We used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-kappaB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-kappaB protein Dif, usually needed for responses against Gram-positive bacteria. CONCLUSIONS/SIGNIFICANCE: Our results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.

Horizontal gene transfer between Wolbachia and the mosquito Aedes aegypti.

BACKGROUND: The evolutionary importance of horizontal gene transfer (HGT) from Wolbachia endosymbiotic bacteria to their eukaryotic hosts is a topic of considerable interest and debate. Recent transfers of genome fragments from Wolbachia into insect chromosomes have been reported, but it has been argued that these fragments may be on an evolutionary trajectory to degradation and loss. RESULTS: We have discovered a case of HGT, involving two adjacent genes, between the genomes of Wolbachia and the currently Wolbachia-uninfected mosquito Aedes aegypti, an important human disease vector. The lower level of sequence identity between Wolbachia and insect, the transcription of all the genes involved, and the fact that we have identified homologs of the two genes in another Aedes species (Ae. mascarensis), suggest that these genes are being expressed after an extended evolutionary period since horizontal transfer, and therefore that the transfer has functional significance. The association of these genes with Wolbachia prophage regions also provides a mechanism for the transfer. CONCLUSION: The data support the argument that HGT between Wolbachia endosymbiotic bacteria and their hosts has produced evolutionary innovation.

Modifying insect population age structure to control vector-borne disease.

Age is a critical determinant of the ability of most arthropod vectors to transmit a range of human pathogens. This is due to the fact that most pathogens require a period of extrinsic incubation in the arthropod host before pathogen transmission can occur. This developmental period for the pathogen often comprises a significant proportion of the expected lifespan of the vector. As such, only a small proportion of the population that is oldest contributes to pathogen transmission. Given this, strategies that target vector age would be expected to obtain the most significant reductions in the capacity of a vector population to transmit disease. The recent identification of biological agents that shorten vector lifespan, such as Wolbachia, entomopathogenic fungi and densoviruses, offer new tools for the control of vector-borne diseases. Evaluation of the efficacy of these strategies under field conditions will be possible due to recent advances in insect age-grading techniques. Implementation of all of these strategies will require extensive field evaluation and consideration of the selective pressures that reductions in vector longevity may induce on both vector and pathogen.

Predicting the age of mosquitoes using transcriptional profiles.

The use of transcriptional profiles for predicting mosquito age is a novel solution for the longstanding problem of determining the age of field-caught mosquitoes. Female mosquito age is of central importance to the transmission of a range of human pathogens. The transcriptional age-grading protocol we present here was developed in Aedes aegypti, principally as a research tool. Age predictions are made on the basis of transcriptional data collected from mosquitoes of known age. The abundance of eight candidate gene transcripts is quantified relative to a reference gene using quantitative reverse transcriptase-PCR (RT-PCR). Normalized gene expression (GE) measures are analyzed using canonical redundancy analysis to obtain a multivariate predictor of mosquito age. The relationship between the first redundancy variate and known age is used as the calibration model. Normalized GE measures are quantified for wild-caught mosquitoes, and ages are then predicted using this calibration model. Rearing of mosquitoes to specific ages for calibration data can take up to 40 d. Molecular analysis of transcript abundance, and subsequent age predictions, should take approximately 3-5 d for 100 individuals.

The use of transcriptional profiles to predict adult mosquito age under field conditions.

Age is a critical determinant of an adult female mosquito's ability to transmit a range of human pathogens. Despite its central importance, relatively few methods exist with which to accurately determine chronological age of field-caught mosquitoes. This fact is a major constraint on our ability to fully understand the relative importance of vector longevity to disease transmission in different ecological contexts. It also limits our ability to evaluate novel disease control strategies that specifically target mosquito longevity. We report the development of a transcriptional profiling approach to determine age of adult female Aedes aegypti under field conditions. We demonstrate that this approach surpasses current cuticular hydrocarbon methods for both accuracy of predicted age as well as the upper limits at which age can be reliably predicted. The method is based on genes that display age-dependent expression in a range of dipteran insects and, as such, is likely to be broadly applicable to other disease vectors.

Benefits of switching from typical to atypical antipsychotic medications: a longitudinal study in a community-based setting.

OBJECTIVE: This study examines the clinical and resource utilization effects of switching stable outpatients with schizophrenia from a typical to an atypical antipsychotic medication. METHOD: We monitored 43 schizophrenia patients from a community mental health program who tolerated switching from typical to atypical antipsychotic medications. We used the Positive and Negative Syndrome Scale (PANSS), Lehman Quality of Life Interview (QOL), and service utilization data for 2 years before and 2 years after the switch. RESULTS: The switch to atypical antipsychotics was associated with significant improvements in positive symptoms, in general psychopathology, and in quality of life. Resource requirements, including case-management and crisis services and hospitalization days, were significantly reduced. We observed no changes in the sample's already low levels of negative symptoms. CONCLUSIONS: In stable outpatients with schizophrenia in a real-world setting, switching to an atypical antipsychotic can result in sustained, significant improvement in clinical response and quality of life, as well as in reduced need for hospitalization and community support.