High-throughput PCR assays to monitor Wolbachia infection in the dengue mosquito (Aedes aegypti) and Drosophila simulans.
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ABSTRACT:
SUBMITTER: Lee SF
PROVIDER: S-EPMC3370494 | biostudies-literature | 2012 Jul
REPOSITORIES: biostudies-literature
Publications
High-throughput PCR assays to monitor Wolbachia infection in the dengue mosquito (Aedes aegypti) and Drosophila simulans.
Lee Siu F SF White Vanessa L VL Weeks Andrew R AR Hoffmann Ary A AA Endersby Nancy M NM
Applied and environmental microbiology 20120420 13
We have developed and validated two new fluorescence-based PCR assays to detect the Wolbachia wMel strain in Aedes aegypti and the wRi and wAu strains in Drosophila simulans. The new assays are accurate, informative, and cost-efficient for large-scale Wolbachia screening. ...[more]
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Project description:BackgroundThe mosquito Aedes aegypti was recently transinfected with a life-shortening strain of the endosymbiont Wolbachia pipientis (wMelPop) as the first step in developing a biocontrol strategy for dengue virus transmission. In addition to life-shortening, the wMelPop-infected mosquitoes also exhibit increased daytime activity and metabolic rates. Here we sought to quantify the blood-feeding behaviour of Wolbachia-infected females as an indicator of any virulence or energetic drain associated with Wolbachia infection.Methodology/principal findingsIn a series of blood-feeding trials in response to humans, we have shown that Wolbachia-infected mosquitoes do not differ in their response time to humans, but that as they age they obtain fewer and smaller blood meals than Wolbachia-uninfected controls. Lastly, we observed a behavioural characteristic in the Wolbachia infected mosquitoes best described as a "bendy" proboscis that may explain the decreased biting success.Conclusions/significanceTaken together the evidence suggests that wMelPop infection may be causing tissue damage in a manner that intensifies with mosquito age and that leads to reduced blood-feeding success. These behavioural changes require further investigation with respect to a possible physiological mechanism and their role in vectorial capacity of the insect. The selective decrease of feeding success in older mosquitoes may act synergistically with other Wolbachia-associated traits including life-shortening and viral protection in biocontrol strategies.
| S-EPMC2734393 | biostudies-literature
Project description:BACKGROUND: Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect. METHODOLOGY/FINDINGS: Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction. CONCLUSIONS/SIGNIFICANCE: This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.
| S-EPMC3738474 | biostudies-literature
Project description:Aedes aegypti transmits one of the most significant mosquito-borne viruses, dengue virus (DENV). The absence of effective vaccines and clinical treatments and the emergence of insecticide resistance in A. aegypti necessitate novel vector control strategies. A new approach uses the endosymbiotic bacterium Wolbachia pipientis to reduce the spread of arboviruses. However, the Wolbachia-mediated antiviral mechanism is not well understood. To shed light on this mechanism, we investigated an unexplored aspect of Wolbachia-virus-mosquito interaction. We used RNA sequencing to examine the transcriptional response of Wolbachia to DENV infection in A. aegypti Aag2 cells transinfected with the wAlbB strain of Wolbachia. Our results suggest that genes encoding an endoribonuclease (RNase HI), a regulator of sigma 70-dependent gene transcription (6S RNA), essential cellular, transmembrane, and stress response functions and primary type I and IV secretion systems were upregulated, while a number of transport and binding proteins of Wolbachia, ribosome structure, and elongation factor-associated genes were downregulated due to DENV infection. Furthermore, bacterial retrotransposon, transposable, and phage-related elements were found among the up- and downregulated genes. We show that Wolbachia elicits a transcriptional response to virus infection and identify differentially expressed Wolbachia genes mostly at the early stages of virus infection. These findings highlight Wolbachia's ability to alter its gene expression in response to DENV infection of the host cell. IMPORTANCE Aedes aegypti is a vector of several pathogenic viruses, including dengue, Zika, chikungunya, and yellow fever viruses, which are of importance to human health. Wolbachia is an endosymbiotic bacterium currently used in transinfected mosquitoes to suppress replication and transmission of dengue viruses. However, the mechanism of Wolbachia-mediated virus inhibition is not fully understood. While several studies have shown mosquitoes' transcriptional responses to dengue virus infection, none have investigated these responses in Wolbachia, which may provide clues to the inhibition mechanism. Our results suggest changes in the expression of a number of functionally important Wolbachia genes upon dengue virus infection, including those involved in stress responses, providing insights into the endosymbiont's reaction to virus infection.
| S-EPMC8265661 | biostudies-literature
Project description: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.
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Project description:MicroRNAs (miRNA) have alternative forms known as isomiRs, which differ from each other by a few nucleotides. Next generation sequencing platforms facilitate identification of these isomiRs and recent discoveries regarding their functional importance have increased our understandings of the regulatory complexities of the microRNAome. Observed changes in the miRNA profiles in mosquitoes infected with flaviviruses have implicated small RNAs in the interactions between viruses and their vectors. Here we analysed the isomiR profiles of both uninfected and infected blood fed Aedes aegypti mosquitoes with a major human pathogen, Dengue virus at two time points post-infection. We found noticeable changes to the isomiR expression profile in response to infection and aging. Data analysis revealed a distinct bias towards isomiR production in the mature miRNA as opposed to the star strand. Furthermore, we noticed that only in 40% of Ae. aegypti miRNAs, the most abundant reads for each particular miRNA match the exact sequence reported in the miRbase. The isomiR expression variations between an Ae. aegypti embryonic cell line (Aag2) and whole mosquitoes demonstrated a tissue-specific pattern of isomiR production. Our results illustrated a bias for certain types of isomiRs for each miRNA. The findings presented in this study also provide evidence that isomiR production is not a random phenomenon and may be important in DENV colonisation of its vector.
2015-11-05 | GSE59516 | GEO
Project description:BackgroundDengue viruses (DENV) are the causative agents of dengue, the world's most prevalent arthropod-borne disease with around 40% of the world's population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia's efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito's ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites.Methodology/principal findingsWe used a non-destructive assay to repeatedly quantify DENV in saliva from wMel-infected and Wolbachia-free wild-type control mosquitoes following the consumption of a DENV-infected blood meal. We show that wMel lengthens the EIP, reduces the frequency at which the virus is expectorated and decreases the dengue copy number in mosquito saliva as compared to wild-type mosquitoes. These observations can at least be partially explained by an overall reduction in saliva produced by wMel mosquitoes. More generally, we found that the concentration of DENV in a blood meal is a determinant of the length of EIP, saliva virus titer and mosquito survival.Conclusions/significanceThe saliva-based traits reported here offer more disease-relevant measures of Wolbachia's effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.
| S-EPMC4482661 | biostudies-literature
Project description:MicroRNAs (miRNA) have alternative forms known as isomiRs, which differ from each other by a few nucleotides. Next generation sequencing platforms facilitate identification of these isomiRs and recent discoveries regarding their functional importance have increased our understandings of the regulatory complexities of the microRNAome. Observed changes in the miRNA profiles in mosquitoes infected with flaviviruses have implicated small RNAs in the interactions between viruses and their vectors. Here we analysed the isomiR profiles of both uninfected and infected blood fed Aedes aegypti mosquitoes with a major human pathogen, Dengue virus at two time points post-infection. We found noticeable changes to the isomiR expression profile in response to infection and aging. Data analysis revealed a distinct bias towards isomiR production in the mature miRNA as opposed to the star strand. Furthermore, we noticed that only in 40% of Ae. aegypti miRNAs, the most abundant reads for each particular miRNA match the exact sequence reported in the miRbase. The isomiR expression variations between an Ae. aegypti embryonic cell line (Aag2) and whole mosquitoes demonstrated a tissue-specific pattern of isomiR production. Our results illustrated a bias for certain types of isomiRs for each miRNA. The findings presented in this study also provide evidence that isomiR production is not a random phenomenon and may be important in DENV colonisation of its vector. Examination of isomiR production rate in DENV infected and non infected mosquitoes
2015-11-05 | E-GEOD-59516 | biostudies-arrayexpress
Project description:INTRODUCTION: Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. METHODOLOGY/PRINCIPAL FINDINGS: Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.
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Project description:A host's immune system plays a central role in shaping the composition of the microbiota and, in return, resident microbes influence immune responses. Symbiotic associations of the maternally transmitted bacterium Wolbachia occur with a wide range of arthropods. It is, however, absent from the dengue and Zika vector mosquito Aedes aegypti in nature. When Wolbachia is artificially forced to form symbiosis with this new mosquito host, it boosts the basal immune response and enhances the mosquito's resistance to pathogens, including dengue, Zika virus and malaria parasites. The mechanisms involved in establishing a symbiotic relationship between Wolbachia and A. aegypti, and the long-term outcomes of this interaction, are not well understood. Here, we have demonstrated that both the immune deficiency (IMD) and Toll pathways are activated by the Wolbachia strain wAlbB upon its introduction into A. aegypti. Silencing the Toll and IMD pathways via RNA interference reduces the wAlbB load. Notably, wAlbB induces peptidoglycan recognition protein (PGRP)-LE expression in the carcass of A. aegypti, and its silencing results in a reduction of symbiont load. Using transgenic mosquitoes with stage-specific induction of the IMD and Toll pathways, we have shown that elevated wAlbB infection in these mosquitoes is maintained via maternal transmission. These results indicate that host innate immunity is utilized to establish and promote host-microbial symbiosis. Our results will facilitate a long-term projection of the stability of the Wolbachia-A. aegypti mosquito system that is being developed to control dengue and Zika virus transmission to humans.
| S-EPMC5739022 | biostudies-literature
Project description:Competition between viruses and Wolbachia for host lipids is a proposed mechanism of Wolbachia-mediated virus blocking in insects. Yet, the metabolomic interaction between virus and symbiont within the mosquito has not been clearly defined. We compare the lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of the Wolbachia wMel strain and dengue virus serotype 3 (DENV3). We found metabolic signatures of infection-induced intracellular events but little evidence to support direct competition between Wolbachia and virus for host lipids. Lipid profiles of dual-infected mosquitoes resemble those of DENV3 mono-infected mosquitoes, suggesting virus-driven modulation dominates over that of Wolbachia. Interestingly, knockdown of key metabolic enzymes suggests cardiolipins are host factors for DENV3 and Wolbachia replication. These findings define the Wolbachia-DENV3 metabolic interaction as indirectly antagonistic, rather than directly competitive, and reveal new research avenues with respect to mosquito × virus interactions at the molecular level.
| S-EPMC7501868 | biostudies-literature