Project description:The Southern house mosquito, Culex quinquefasciatus, is a vector of the causative agents of many diseases including West Nile Fever, St. Louis Encephalitis, and lymphatic filariasis. In order to manage the spread of these diseases, vector control efforts rely heavily on insecticides, including pyrethroids, namely permethrin. In our study we investigated the changes in the gene expression profiles of a highly-permethrin resistant strain of Cx. quinquefasciatus during constant exposure to permethrin at the LC-0, LC-50, and LC-70 rates, which killed 0, 50, and 70% of all larvae, respectively. Overall, we identified several genes that were up-regulated including detoxification genes such as cytochrome P450s as well as genes that were down-regulated.
Project description:Culex pipiens molestus and Cx. p. quinquefasciatus are the members of Culex pipiens Complex, but they display relatively large differences in behavior and physiological responses. We compared the genes of these mosquitoes to identify those that were differentially expressed in each subspecies. Such genes could play important roles in subspecies-specific blood feeding or oviposition behavior. Culex pipiens molestus and Cx. p. quinquefasciatus females were undertaken Illumina RNA sequencing.
Project description:Mosquito-borne flaviviruses maintain life cycles in mammals and mosquitoes. RNA interference (RNAi) has been demonstrated as an anti-flavivirus mechanism in mosquitoes; however, whether and how flavivirus induces and antagonizes RNAi-mediated antiviral immunity in mammals remains unknown. Here we showed that NS2A of Dengue virus-2 (DENV2) act as a viral suppressor of RNAi (VSR). When NS2A-mediated RNAi suppression was disabled, the resulting mutant DENV2 induced Dicer-dependent production of abundant DENV2-derived siRNAs in differentiated mammalian cells. Importantly, VSR-disabled DENV2 showed severe replication defects in mosquito and mammalian cells, and mice, which were rescued by the deficiency of RNAi. Moreover, NS2As of multiple flaviviruses act as VSRs in vitro and during viral infection in both organisms. Overall, our findings demonstrate that antiviral RNAi can be induced by flavivirus, while flavivirus uses NS2A as bona fide VSR to evade RNAi in mammals and mosquitoes, highlighting the importance of RNAi in flaviviral vector-host life cycles.
Project description:RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we showed that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and Cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived siRNA into the RNA-induced silencing complex, thereby enhancing antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited an RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited replication of flaviviruses, including Dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrated that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control vector transmission of arboviruses or viral diseases in insect farming.
Project description:Culex pipiens pallens and Cx. p. quinquefasciatus are important vectors of many diseases, such as West Nile fever and lymphatic filariasis. The widespread use of insecticides to control these disease vectors and other insect pests has led to insecticide resistance becoming common in these species. High throughput screening using SSH and specific microarray platforms was thought to have identified some resistance-related genes. However, limitations of these methods meant that only a few hundred of the many thousand genes could be screened. It wasn’t until the sequencing of the Cx. quinquefasciatus genome in 2010 that it became possible to screen all 18.9 thousand genes in the mosquito genome for anti-insecticidal activity. We used high throughout Illumina sequencing to identify hundreds of Cx. p. pallens and Cx. p. quinquefasciatus genes that were differentially expressed in response to pesticide exposure. The identification of these genes is a vital first step for more detailed investigation of the molecular mechanisms involved in insecticide resistance in mosquitoes. In this study, larvae of Cx. pipiens pallens and Cx. pipiens quinquefasciatus were collected from field and transported to the laboratory and reared to adulthood to get F1 generation. Then, half of the F1 generation was conducted to pesticide bioassay. RNA extraction and Illumina sequencing were undertaken in another half of the F1 generation. Therefore, Samples used in Illumina sequencing did not contact any insecticides. Twelve Cx. pipiens pallens and Cx. pipiens quinquefasciatus lavae were undertaken Illumina RNA sequencing.
Project description:The genomes of three major mosquito vectors of human diseases, including Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been previously sequenced. C. p. quinquefasciatus has the largest number of predicted protein-coding genes, which partially results from the expansion of three detoxification gene families: cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), and carboxylcholinesterases (CCE). However, unlike A. gambiae and A. aegypti, which have large amounts of gene expression data, C. p. quinquefasciatus has limited transcriptomic resources. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes. In the present study, the three detoxification gene families of C. p. quinquefasciatus were analyzed for phylogenetic classification and compared with those of three other dipteran insects. Gene expression during various developmental stages and the differential expression responsible for parathion resistance were profiled using the digital gene expression (DGE) technique. Results: A total of 291 detoxification genes were found in C. p. quinquefasciatus, including 70 CCE, 186 P450, and 35 GST genes. Compared with three other dipteran species, gene expansion in Culex mainly occurred in the CCE and P450 families, where the genes of M-NM-1-esterases, juvenile hormone esterases, and CYP325 of the CYP4 subfamily showed the most pronounced expansion on the genome. A total of 13314 genes were expressed in five DGE libraries. Genes with signal transduction and odorant binding functions were prominently expressed during egg development. Genes involved in proteolysis, glycosphingolipid biosynthesis, and purine metabolism were preferentially expressed at the larval stage. Seventy five percent of the detoxification genes were found to be expressed. One fourth of the CCE and P450 genes were expressed at unique stages, indicating their developmentally regulated expression. Fifteen detoxification genes, including 2 CCEs, 6 GSTs, and 7 P450s, were expressed at higher levels in a parathion-resistant strain than in a susceptible strain. Conclusion: The results of the present study provide new insights into the functions and evolution of three detoxification gene families in mosquitoes and comprehensive transcriptomic resources for C. p. quinquefasciatus, which will facilitate the elucidation of molecular mechanisms underlying the different biological characteristics of the three major mosquito vectors. Raw data were deposited in SRA and assigned accession number SRA049959: http://www.ncbi.nlm.nih.gov/sra?term=SRA049959 Five DGE libraries were sequenced: the egg, third instar larval, pupal, and adult stages of the SG strain, and the third instar larval stage of the S-lab strain.
Project description:Lysinibacillus sphaericus produces the mosquito larvicidal binary toxin consisting of BinA and BinB, which are both required for toxicity against Culex and Anopheles larvae. The molecular mechanisms behind Bin toxin-induced damage remain unexplored. We used whole-genome microarray-based transcriptome analysis to better understand how Culex larvae respond to Bin toxin treatment at the molecular level. Our analyses of Culex quinquefasciatus larvae transcriptome changes at 6, 12, and 18 h after Bin toxin treatment revealed a wide range of transcript signatures, including genes linked to the cytoskeleton, metabolism, immunity, and cellular stress, with a greater number of down-regulated genes than up-regulated genes. Bin toxin appears to mainly repress the expression of genes involved in metabolism, the mitochondrial electron transport chain, and the protein transporter of the outer/inner mitochondrial membrane. The induced genes encode proteins linked to mitochondrial-mediated apoptosis and cellular detoxification including autophagic processes and lysosomal compartments.
Project description:Analysis of Culex quinquefasciatus responses to West Nile virus (WNV) infection at 7 and 14 days after ingestion of infected blood in the gut and carcass tissues.
Project description:The Southern house mosquito, Culex quinquefasciatus, is an anautogenous mosquito species that requires a blood meal in order to provision the eggs. Following the eclosion of the adults from the pupal stage, adult female mosquitoes require a period of time for mating and development before they are competent to take a blood meal. In order to better understand the genes involved in preparing the females to take a blood meal, populations of non-blooded adult female Cx. quinquefasciatus were collected from even-aged populations and used for RNA Seq analysis. A total of seven post-eclosion time points were selected (2, 12, 24, 36, 48, 60, and 72 hours), which spanned the pre-blood feeding time period and the time period during which the females were competent for the acquisition of the blood meal. Overall, the majority of differentially-expressed genes were identified between the 2 and 12h time points with most genes reaching stable expression after 36h. This study identified the global changes in gene expression profiles over time as the females become competent to acquire the blood meal.