Gene expression profiling in wMelPop-infected Aedes aegypti
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ABSTRACT: Whole genome transcriptional profiling comparing Ae. aegypti infected with Wolbachia sp. wMelPop (PGYP1) to an uninfected Ae. aegypti control line (PGYP1.tet). The objective of the experiment was to identify genes that may be involved in the life shortening phenotype associated with wMelPop infection. Two-colour experiment; infected vs. uninfected Ae. aegypti; 4 biological replicates; 2 dye swaps.
Project description:Whole genome transcriptional profiling comparing Ae. aegypti infected with Wolbachia sp. wMelPop (PGYP1) to an uninfected Ae. aegypti control line (PGYP1.tet). The objective of the experiment was to identify genes that may be involved in the life shortening phenotype associated with wMelPop infection.
Project description:The incomplete genome annotation of non-model organisms hampers molecular and proteomic studies. Proteomics informed by transcriptomics (PIT) is suited to non-model organisms because peptides are identified using transcriptomic, not genomic, data. Aedes aegypti is the mosquito vector for the (re-)emerging dengue, chikungunya, yellow fever and Zika viruses. An Ae. aegypti genome sequence is available, however experimental evidence for >90% of the Ae. aegypti proteome or the activity of transposable elements (TEs) that constitute 50% of the Ae. aegypti genome is lacking. We used PIT to characterise the proteome of the Aedes aegypti derived cell line Aag2. Hotspots of incomplete genome annotation were identified which are not explained by poor sequence and assembly quality. We developed criteria for the characterisation of proteomically active TEs and demonstrate that protein expression does not correlate with a TE’s genomic abundance. Finally, we identify Phasi Charoen-like virus as an unrecognised contaminant of Aag2 cells. We therefore present the first proteomic characterisation of mobile genetic elements, and provide proof-of-principle that PIT can evaluate a genome’s annotation to guide annotation efforts.
Project description:Wolbachia pipientis is an intracellular symbiotic bacterium found in insects and arthropods. Wolbachia can decrease the vectorial capacity for various pathogens, such as the dengue virus, in Aedes aegypti. The purpose of this study was to determine the effect of Wolbachia (wMel strain) on the vectorial capacity of Ae. aegypti for Dirofilaria immitis. We analyzed gene expression patterns by RNA-seq in addition to the D. immitis infection phenotype in Ae. aegypti infected with and without wMel. Four Ae. aegypti strains, MGYP2.tet, MGYP2, Liverpol (LVP)-Obihiro (OB), and LVP-OB-wMel (OB-wMel) were analyzed for transcriptome comparison in Malpighian tubule at 2 days post infection. The correlation between Wolbachia infection, D. immitis infection phenotype and immune-related genes expression in Ae. aegypti was investigated.
Project description:Dengue viruses (DENV) are generally maintained in a cycle which requires horizontal transmission via their arthropod vector, Ae. Aegypti, to the vertebrate host. One important consequence of this process is the interference of these arboviruses with both invertebrate and vertebrate immune systems. While infection of vertebrates causes disease, the presence of DENV gives rise to life-long, persistent infection in mosquitoes. The results of a comparative transcriptome analysis between DENV-infected and uninfected salivary glands revealed activation of both the immune deficiency (IMD) and the Toll pathways, as well as involvement of the putative antibacterial cecropin-like peptide (AAEL000598), in controlling DENV infection in Ae. aegypti. The mature form of this peptide was found to be active against DENV and Chikungunya viruses, whereas its precursor also had a strong anti-Leishmania effect. This study is the first to establish a comparative transcriptome analysis of DENV-infected and uninfected salivary glands and demonstrates that certain DENV-induced peptides, that are part of the IMD pathway, possess broad-spectrum anti-pathogenic activity and may have therapeutic potential in human.
Project description:Background: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it be responsible for transposon silencing in this mosquito. Results: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. Conclusions: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster.
Project description:Dengue viruses (DENV) are generally maintained in a cycle which requires horizontal transmission via their arthropod vector, Ae. Aegypti, to the vertebrate host. One important consequence of this process is the interference of these arboviruses with both invertebrate and vertebrate immune systems. While infection of vertebrates causes disease, the presence of DENV gives rise to life-long, persistent infection in mosquitoes. The results of a comparative transcriptome analysis between DENV-infected and uninfected salivary glands revealed activation of both the immune deficiency (IMD) and the Toll pathways, as well as involvement of the putative antibacterial cecropin-like peptide (AAEL000598), in controlling DENV infection in Ae. aegypti. The mature form of this peptide was found to be active against DENV and Chikungunya viruses, whereas its precursor also had a strong anti-Leishmania effect. This study is the first to establish a comparative transcriptome analysis of DENV-infected and uninfected salivary glands and demonstrates that certain DENV-induced peptides, that are part of the IMD pathway, possess broad-spectrum anti-pathogenic activity and may have therapeutic potential in human. Infectious blood meals were offered to 3-day-old, adult, female Ae. aegypti Liverpool mosquitoes using a silicone membrane feeder system (Alto et al., 2005). Human blood was combined with DENV-2 16681 to provide a blood meal titer of 5.106 plaque forming units (PFU)/ml. At different time-points after the blood meal, salivary glands were dissected in acid guanidium thiocyanate-phenol-chloroform (RNAble; Eurobio, France) or phosphate-buffered saline (PBS), and the samples were frozen at -70°C until use.
Project description:Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida Ae. aegypti in response to Zika virus infection. A total of 2,459 transcripts were expressed at significantly different levels between resistant and susceptible Ae. aegypti. Gene ontology analysis placed these genes into 7 categories of biological processes. The 863 transcripts were expressed at significantly different levels between two strains (up/down regulated) more than 2-fold. Quantitative real-time PCR analysis validated Zika-infected response, and suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the molecular level allows for the possible development of new approaches in mitigating arbovirus transmission. This information sheds light on Zika-induced changes in the insecticide resistance of Ae. aegypti with implications for mosquito control strategies.
Project description:We have generated transgenic Ae. aegypti that express a ZIKV-specific inverted repeat RNA intended to trigger the mosquito siRNA anti-viral immune pathway. To confirm that the transgene is expressed and processed, we performed small RNA sequencing on both Higgs White Eye (HWE) & anti-NS3/4A-ZIKV transgenic Ae. aegypti 24 hours post-bloodmeal
Project description:Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example Guillain-Barré syndrome. One of the actual most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. In order to investigate the impact of co-infections on the reproductive tract of the mosquito, we applied a quantitative proteomic strategy based on an isobaric labeling to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. For what we know, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3913 proteins identified. We were able to quantify a total of 1044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary employing mass spectrometry-based quantitative methods. Furthermore, we describe and discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, co-infection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti.
Project description:Background: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it be responsible for transposon silencing in this mosquito. Results: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. Conclusions: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster. 5 small RNA libraries were generated from total RNA of whole adult Aedes aegypti tissues, two of these libraries were sequenced twice (technical replicates). 1 small RNA library was generated from total RNA of a whole adult Drosophila melanogaster tissue.