RESUMO
Juvenile hormone (JH) controls the development and reproduction of insects. Therefore, a tight regulation of the expression of JH biosynthetic enzymes is critical. microRNAs (miRNAs) play significant roles in the post-transcriptional regulation of gene expression by interacting with complementary sequences in target genes. Previously, we reported that several miRNAs were differentially expressed during three developmental stages of Aedes aegypti mosquitoes with different JH levels (no JH, high JH, and low JH). One of these miRNAs was aae-miR-34-5p. In this study, we identified the presence of potential target sequences of aae-miR-34-5p in the transcripts of some genes encoding JH biosynthetic enzymes. We analysed the developmental expression patterns of aae-miR-34-5p and the predicted target genes involved in JH biogenesis. Increases in miRNA abundance were followed, with a delay, by decreases in transcript levels of target genes. Application of an inhibitor and a mimic of aae-miR-34-5p led respectively to increased and decreased levels of thiolase transcripts, which is one of the early genes of JH biosynthesis. Female adult mosquitoes injected with an aae-miR-34-5p inhibitor exhibited significantly increased transcript levels of three genes encoding JH biosynthetic enzymes, acetoacetyl-CoA thiolase (thiolase), farnesyl diphosphate phosphatase, and farnesal dehydrogenase. Overall, our results suggest a potential role of miRNAs in JH production by directly targeting genes involved in its biosynthesis.
Assuntos
Aedes , MicroRNAs , Animais , Feminino , Hormônios Juvenis/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
The endosymbiotic bacterium Wolbachia pipientis blocks replication of several arboviruses in transinfected Aedes aegypti mosquitoes. However, the mechanism of virus blocking remains poorly understood. Here, we characterized an RNase HI gene from Wolbachia, which is rapidly induced in response to dengue virus (DENV) infection. Knocking down w RNase HI using antisense RNA in Wolbachia-transinfected mosquito cell lines and A. aegypti mosquitoes led to increased DENV replication. Furthermore, overexpression of wRNase HI, in the absence of Wolbachia, led to reduced replication of a positive sense RNA virus, but had no effect on a negative sense RNA virus, a familiar scenario in Wolbachia-infected cells. Altogether, our results provide compelling evidence for the missing link between early Wolbachia-mediated virus blocking and degradation of viral RNA. These findings and the successful pioneered knockdown of Wolbachia genes using antisense RNA in cell line and mosquitoes enable new ways to manipulate and study the complex endosymbiont-host interactions.
RESUMO
Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa.
Assuntos
Aedes/metabolismo , MicroRNAs/sangue , Mosquitos Vetores/metabolismo , Vitelogeninas/metabolismo , Aedes/citologia , Aedes/genética , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Corpo Adiposo/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas de Insetos/metabolismo , MicroRNAs/genética , Mosquitos Vetores/genética , Proteômica/métodos , RNA-Seq/métodos , Espectrometria de Massas em Tandem/métodos , Vitelogeninas/genéticaRESUMO
The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This "pathogen blocking" could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV), a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2-5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking.
Assuntos
Aedes , Colesterol/farmacologia , Dicistroviridae/metabolismo , Gorduras na Dieta/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Wolbachia/fisiologia , Aedes/metabolismo , Aedes/microbiologia , Aedes/virologia , Animais , Colesterol/metabolismo , Gorduras na Dieta/metabolismo , Drosophila melanogaster , Interações Hospedeiro-Patógeno/fisiologiaRESUMO
Wolbachia pipientis delays RNA virus-induced mortality in Drosophila spp. We investigated whether Wolbachia-mediated protection was dependent on the small interfering RNA (siRNA) pathway, a key antiviral defense. Compared to Wolbachia-free flies, virus-induced mortality was delayed in Wolbachia-infected flies with loss-of-function of siRNA pathway components, indicating that Wolbachia-mediated protection functions in the absence of the canonical siRNA pathway.
Assuntos
Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Redes e Vias Metabólicas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Wolbachia/genética , Wolbachia/metabolismo , Animais , Drosophila melanogaster/imunologia , Vírus de RNA/imunologia , Análise de SobrevidaRESUMO
The outcome of microbial infection of insects is dependent not only on interactions between the host and pathogen, but also on the interactions between microbes that co-infect the host. Recently the maternally inherited endosymbiotic bacteria Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens. Mosquitoes experimentally infected with Wolbachia have upregulated immune responses and are protected from a number of pathogens including viruses, bacteria, Plasmodium and filarial nematodes. It has been hypothesised that immune upregulation underpins Wolbachia-mediated protection. Drosophila is a strong model for understanding host-Wolbachia-pathogen interactions. Wolbachia-mediated antiviral protection in Drosophila has been demonstrated for a number of different Wolbachia strains. In this study we investigate whether Wolbachia-infected flies are also protected against pathogenic bacteria. Drosophila simulans lines infected with five different Wolbachia strains were challenged with the pathogenic bacteria Pseudomonas aeruginosa PA01, Serratia marcescens and Erwinia carotovora and mortality compared to paired lines without Wolbachia. No difference in mortality was observed in the flies with or without Wolbachia. Similarly no antibacterial protection was observed for D. melanogaster infected with Wolbachia. Interestingly, D. melanogaster Oregon RC flies which are naturally infected with Wolbachia showed no upregulation of the antibacterial immune genes TepIV, Defensin, Diptericin B, PGRP-SD, Cecropin A1 and Attacin D compared to paired flies without Wolbachia. Taken together these results indicate that Wolbachia-mediated antibacterial protection is not ubiquitous in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent. We suggest that the immune priming and antibacterial protection observed in Wolbachia-infected mosquitoes may be a consequence of the recent artificial introduction of the symbiont into insects that normally do not carry Wolbachia and that antibacterial protection is unlikely to be found in insects carrying long-term Wolbachia infections.
Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Wolbachia/imunologia , Animais , Drosophila melanogaster/genética , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Wolbachia/efeitos dos fármacos , Wolbachia/crescimento & desenvolvimento , Wolbachia/patogenicidadeRESUMO
Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.
Assuntos
Aedes/microbiologia , Vírus Chikungunya/fisiologia , Vírus da Dengue/fisiologia , Plasmodium gallinaceum/fisiologia , Wolbachia/fisiologia , Aedes/parasitologia , Aedes/fisiologia , Aedes/virologia , Animais , Interações Hospedeiro-Parasita , SimbioseRESUMO
Wolbachia pipientis bacteria are common endosymbionts of insects that are best known for their ability to increase their prevalence in populations by manipulating host reproductive systems. However, there are examples of Wolbachia that exist in nature that seem to induce no reproductive parasitism trait and yet are able to invade populations. We demonstrate a fitness benefit for Wolbachia-infected insects that may explain this paradox. Drosophila melanogaster flies infected with Wolbachia are less susceptible to mortality induced by a range of RNA viruses. The antiviral protection associated with Wolbachia infection might be exploited in future strategies to reduce transmission of pathogens by insects.
Assuntos
Drosophila melanogaster/microbiologia , Drosophila melanogaster/virologia , Vírus de Insetos/patogenicidade , Vírus de RNA/patogenicidade , Wolbachia/fisiologia , Animais , Drosophila melanogaster/fisiologia , Interações Hospedeiro-Patógeno , Nodaviridae/patogenicidadeRESUMO
Insect responses that are specific for virus infection have been investigated using the genetically tractable Drosophila melanogaster. Most studies focus on interactions with Drosophila C virus (DCV), which is a member of the family Dicistroviridae. DCV is a non-enveloped, T=3 icosahedral virus with a positive-sense RNA genome. It was demonstrated recently that several genes controlled by the Jak-STAT pathway are specifically upregulated upon DCV infection. To investigate the virus factors that induce these responses, we used the Jak-STAT regulated genes as reporter genes. Challenge of flies with non-infectious DCV particles or double-stranded RNA did not stimulate significant upregulation of the antiviral response genes. In addition, there was no difference in reporter gene upregulation between Drosophila challenged with three different strains of DCV. This suggests that upregulation of these Drosophila genes may require virus replication and may involve the non-structural proteins of DCV.