Progress 01/01/12 to 05/10/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:PD is no longer at Virginia Tech, nothing to report. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
n/a
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Entomologists, public health professionals, CDC. Changes/Problems:The PD has left the University, no further research or information on this project will be submitted. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The PD left the University, and the project is to be terminated.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Entomologists, public health professionals, CDC. Changes/Problems:PD will be leaving Virginia Tech in the summer of 2016. What opportunities for training and professional development has the project provided?During the performance period, I served as a mentor to three PhD candidate and one M.S. student. One of these students was an active participant in the research described above and gained training in molecular biology and genetics research. How have the results been disseminated to communities of interest?Dissemination of the project activities occurred through publication of results as well as presentations at several invited seminars across the country. Additionally, knowledge about our activities was disseminated through interactions with public school instructors and high school level students, as my students spoke about the experiments and methods they were utilizing to groups of high school level students during outreach activities organized by theLife Sciences Fralin Institute. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Continue to characterize newly generated null mutant mosquito strains for their effect on RNA silencing. Objective 2: Write manuscript and submit completed results; no further experiments. Objective 3: Complete QTL analysis upon receiving final sequencing data; write manuscript and submit.
Impacts
What was accomplished under these goals?
Objective 1- We have continued our focus on double-stranded RNA binding proteins that appear to be involved in exo-siRNA, endo-siRNA and miRNA pathways in the mosquito; these proteins are thought to be important regulators of viral infection. After publishing our cell culture findings during the reporting period, we have begun to generate loss-of-function deletion mutants for a series of target genes suspected of contributing to the anti-viral RNAi response in mosquitoes. In addition to the double-stranded RNA binding proteins we described previously (R2D2, loqs-A, Loqs-B), we have used the CRISPR-Cas9 system to generate deletions in genes VIG and FXR. We were unable to recover mutations in HEN-1. Phenotypic analysis of the mutant strains is ongoing. For the new gene exloqs we identified previously, we successfully generated null mutants which we have employed to examine phenotypic differences between exloqs mutant individuals and their heterozygous siblings. High-throughput sequencing revealed a small group of genes whose expression is severely altered in exloqs mutant mosquitoes. We are continuing to analyze this gene through protein-protein interactions and sequencing of small RNAs. Finally, we have performed a yeast 2-hybrid screen and identified novel interacting factors with loqs-A. Additional screens will be performed with the other proteins. Objective 2- Consistent with what we observedwhen we compared Aedes aegypti and Aedes albopictus, the Aedes hensilli comparison revealed that critical RNAi components AGO2, DCR2 and R2D2 are not rapidly evolving amongst Aedes spp. All three were again found to be under strong purifying selection, with rates of nucleotide substitutions consistent with the 50th percentile of genes in the genome. Thus, we find no evidence of rapid, positive selection among known components of the RNAi pathway. In collaboration with the Fairfax Department of Public Health, we have obtained several hundred wild-caught mosquitoes each for the following species: Ae. vexans, Ae. (Oc.) triseriatus, Ae. (Oc.) japonicus, and Ae. (Oc.) canadensis. Evolutionary comparisons with these mosquitoes are ongoing, but have confirmed our initial findings. Objective 3- After regenerating our transgenic RNAi-reporting sensor strain and generating two homozygous strains, we established a temperature regimen to better differentiate bright (poor RNAi) from dim (strong RNAi) individuals. Five generations of selection have been performed and we have completed selection of strong-silencing and weak-silencing strains. RNAseq analysis of the poor-RNAi and strong-RNAi strains revealed several hundred genes whose expression had changed. Analysis of single nucleotide polymorphisms in the resultant data revealed several candidate genes potentially disrupted in the poor-RNAi strain. Three replicate crosses between poor-RNAi and strong RNAi strains have been performed, with F1 progeny self-crossed. F2 progeny were screened for the bright and dim phenotype and harvested for pooled DNA analysis. We have constructed DNA libraries and now await the final dataset and QTL analysis to identify the genome regions responsible for the silencing phenotypes.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audiences for this project are undergraduate and graduate level students, as well as the greater scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? During the performance period, I served as a mentor to one PhD candidate and one M.S. student. These students were active participants in the research described above and gained training in molecular biology and genetics research. How have the results been disseminated to communities of interest? Dissemination of the project activities occurred through publication of results as well as presentations at several invited seminars across the country. Additionally, knowledge about our activities was disseminated through interactions with public school instructors and high school level students, as my students spoke about the experiments and methods they were utilizing to groups of high school level students during outreach activities organized by the Fralin Institute for Life Sciences. What do you plan to do during the next reporting period to accomplish the goals? Determination of genes which positively or negatively regulate the RNA interference pathway in Ae. aegypti. We have described the generation and validation of a transgenic strain of Aedes aegypti that "senses" the status of the RNAi pathway. White-eyed "sensor" Ae. aegypti express enhanced green fluorescent protein (EGFP), as well as dsRNA homologous to a portion of the EGFP gene in the eye of the mosquito. Under standard conditions, transgenic "sensor" mosquitoes exhibit little EGFP expression, indicating efficient RNA silencing. When a gene product important for RNAi is knocked-down, significant increases in EGFP expression are observed. Using this transgenic "sensor" strain we proposed to systematically test and characterize mosquito genes for involvement in RNAi. Determination of selection pressures acting on putative RNAi genes within the genus Aedes. Work in Drosophila has shown that several genes related to antiviral RNAi are among the fastest evolving in the fly. Demonstrating that the mosquito RNAi pathway is undergoing rapid adaptive evolution, a signature of host-pathogen arms races, could suggest a mechanism for the generation of previously observed inter- and intra-species differences in vector competence and vectorial capacity. In addition, disease vector mosquitoes encounter many diverse human viral pathogens never seen by the fruit fly immune system, and how these viruses shape the evolution of RNAi genes is not known. Although at present, genome sequences for three major disease vectors are available (Anopheles, Aedes and Culex), the large evolutionary distances between them makes investigations into adaptive evolution unfeasible. We proposed to obtain transcriptome data for the disease vectors Ae. albopictus and Ae. hensilli and to perform evolutionary comparisons among RNAi genes with the closely related Ae. aegypti. We proposed to determine whether adaptive forces such as positive or purifying selection are acting on RNAi genes. RNAi genes under positive selection might be interacting most directly with viral pathogens and thus could be the focus of future virus-vector interaction studies or intervention strategies. Determination of the role of genetic variability in RNAi and arbovirus infection. Mosquitoes are naturally exposed to a diverse array of viral pathogens (both human and non-human) in varying frequencies in different geographic areas. Genetic variation in RNAi pathways could help explain some of the differences in vector competence and vectorial capacity that have been previously observed for YFV, CHIKV and DENV. Because vector competence appears to be a complex and multifactorial trait, it has been difficult to effectively measure the contribution of any single mechanism or pathway to this trait. Further, even in model organisms, the role of genetic variation on RNAi and virus defense has not been explicitly tested or explored. Using our transgenic "sensor" strain, which exhibits variation in the strength of RNA silencing from generation to generation, we proposed to directly explore the role of allelic variation in RNAi genes in determining the competence of mosquitoes for CHIKV, DENV and YFV.
Impacts
What was accomplished under these goals?
Aim1- We have continued our focus on double-stranded RNA binding proteins that appear to be involved in antiviral defence pathways in the mosquito. Most strikingly are our observations that these proteins appear to behave quite distinctly in Aedes mosquitoes from their Drosophila (fruit fly) counterparts. We have obtained convincing evidence that the protein AeR3D1-A, unlike DmR3D1-A, is involved in gene silencing and can interact with RNAi components as well as miRNA components. This suggests fundamental differences between mosquitoes and flies as to how these two silencing pathways are regulated, and thus how they might defend themselves against viral replication. We also observed that AeR3D1-A protein appeared to alter its interaction preferences upon viral infection. In uninfected cells, AeR3D1-A interacted strongly with both AGO1 and AGO2; interactions with DCR1 and DCR2 were weak to undetectable. However, in the presence of Sindbis virus (SINV), AeR3D1-A interacted strongly with DCR1 and DCR2, with a reduced affinity for AGO proteins. Small RNA sequencing confirmed that AeR2D2 and AeR3D1-A are involved in exo- and endo-siRNA based silencing while AeR3D1-B controls miRNAs. For the new gene exloqs we identified previously, we successfully generated null mutants which we have employed to examine phenotypic differences between exloqs mutant individuals and their heterozygous siblings. Since we had previously shown exloqs expression is largely restricted to the germline, we analyzed the number of viable progeny produced by each group. Exloqs mutant female mosquitoes produced 15-30% fewer viable progeny than heterozygous females (P<0.0001, Fishers Exact test). Thus, Exloqs protein is critical for full fertility of Aedes aegypti mosquitoes. High-throughput sequencing revealed a small group of genes whose expression is severely altered in exloqs mutant mosquitoes. Of these, one prime candidate is involved in iron homeostasis and may explain the differences in survival. Aim2- Consistent with what we observed in the when we compared Aedes aegypti and Aedes albopictus, the Aedes hensilli comparison revealed that critical RNAi components AGO2, DCR2 and R2D2 are not rapidly evolving amongst Aedes spp. All three were again found to be under strong purifying selection, with rates of nucleotide substitutions consistent with the 50th percentile of genes in the genome. Thus, we find no evidence of rapid, positive selection among known components of the RNAi pathway. In collaboration with the Fairfax Department of Public Health, we have obtained several hundred wild-caught mosquitoes each for the following species: Ae. vexans, Ae. (Oc.) triseriatus, Ae. (Oc.) japonicus, and Ae. (Oc.) canadensis. Evolutionary comparisons with these mosquitoes are ongoing. Aim3- In past years, we experienced substantial problems with generating a homozygous transgenic sensor strain due to inbreeding effects and sex linkage. In the past year, we have established 6 new transgenic sensor strains in a more robust genetic background. Two of these have been made homozygous and we have established a temperature regimen to better differentiate bright (poor RNAi) from dim (strong RNAi) individuals. Five generations of selection have been performed and we have completed selection of strong-silencing and weak-silencing strains. We are now beginning crosses for QTL analysis to identify the genome regions responsible for the silencing phenotypes.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Target audiences for this project are undergraduate and graduate level students, as well as the greater scientific community. Efforts included outreach to K-12 students and teachers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? During the performance period, I served as a mentor to two PhD candidate students. These students were active participants in the research described above and gained training in molecular biology and genetics research; both successfully defended their dissertations in 2013. How have the results been disseminated to communities of interest? Dissemination of the project activities occurred through presentations at the annual meeting of the American Society of Tropical Medicine and Hygiene, the Arthropod Genomics Symposium, and the International Workshop on Transgenesis and Genomes of Invertebrate Organisms, as well as through interactions and interviews with the press following several high profile publications. Additionally, knowledge about our activities was disseminated through interactions with public school instructors and high school level students, as my students spoke about the experiments and methods they were utilizing to groups of high school level students during outreach activities organized by the Fralin Institute for Life Sciences. What do you plan to do during the next reporting period to accomplish the goals? Aim 1: For this objective, we plan to finish characterization of all the genes mentioned in Table 3 of the original proposal. We have already examined most of these in our transgenic sensor strain, and are in the process of performing additional replications to increase statistical power. Most interestingly, the evolutionary analysis performed as part of specific aim 2 has revealed several potential RNAi genes undergoing rapid positive selection. We will be prioritizing study of these genes and their true role in RNA interference in the coming year. Aim 2: The only remaining work for this section is the evolutionary comparison of our newly assembled Ae. hensilli sequence data with those of Ae. aegypti and Ae. albopictus. This comparison will allow us to determine those genes under selection amongst all three species, and those being selected specifically in just one of these. We anticipate submitting a completed manuscript summarizing all of the results of this aim during the current year. If time and funds permit, we will also seek to expand our analysis to additional Aedes species. For instance, we have been in contact with individuals conducting mosquito surveillance who have agreed to provide additional species including Aedes vexans, Ae. (Oc.) japonicus, and Ae. (Oc.) triseriatus. Aim 3: In the coming year, we will infect bright and dim sensor strains with various arboviruses (SINV, CHIKV, and/or YFV) and determine the correlation between silencing of EGFP and susceptibility to viral infection as proposed in our application. One limitation of these experiments is that we are limited to the genetic variability present in the khw strain of Ae. aegypti, which has been in colony for >70 years. We have obtained a field strain of Ae. aegypti collected in Uganda that is only about 10 generations removed from the field. While some loss of genetic diversity is inevitable, we expect this strain to be far more diverse genetically. We will introduce our sensor transgene into this additional strain, and perform the same experiments; correlating viral infection with EGFP silencing phenotypes.
Impacts
What was accomplished under these goals?
Aim 1- We have examined the role of most of the genes identified in our application as being potentially involved in RNA interference in mosquitoes. In addition to the transgenic sensor we described in our application, we have also developed and validated sensors based on the endo-siRNA, exo-siRNA and miRNA pathways. Performing gene knockdown experiments has allowed us to determine not just the participants in the RNAi pathway, but the subpathway and the degree of overlap as well. In addition to identifying components of the RNAi pathway, we also proposed to perform molecular characterization of the participating gene products. In this regard, we have probed the cellular localization and protein-protein interactions between several double-stranded RNA binding proteins and the core components of the RNAi machinery. Aim 2- We have completed transcriptome sequencing for two additional mosquito species: Aedes albopictus and Aedes hensilli. Evolutionary comparisons have revealed selection pressures acting between these species, and we are following up with functional characterization of the genes presumed to be involved in anti-viral immunity that appear to be under the strongest selective pressure. Aim 3- The two sensor strains we had previously published could not be made homozygous due to inbreeding effects. We have generated six new transgenic strains expressing the same construct, several of which we have found successfully silence the transgene. We are in the process of generating homozygous strains to begin our proposed selection experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Aryan, A., Anderson, M. A. E., Myles, K. M., and Adelman Z. N. (2013) TALEN-based gene disruption in the dengue vector Aedes aegypti. PLoS One 8 (3): e60082.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Output activities performed during the project period consisted primarily of conducting laboratory-based experiments and analyzing the resultant data. These experiments included analyzing the function of novel promoter sequences in transgenic mosquitoes, cloning and sequencing of mosquito genes involved in the RNAi pathway, and the generation of various genetically modified strains of mosquito. During the performance period, I served as a mentor to two PhD candidate students. These students were active participants in the research described above, and gained training in molecular biology and genetics research. Dissemination of the project activities occurred through presentations at the annual meeting of the Entomological Society of America, as well as through interactions with public school instructors and high school level students, as my students spoke about the experiments and methods they were utilizing to groups of high school level students during outreach activities organized by the Fralin Institute for Life Sciences. This served the dual purpose of providing training for graduate level students in presenting their work, but also in disseminating knowledge to K-12 students. PARTICIPANTS: Ph.D. Entomology Graduate Students Azadeh Aryan and Mary Etna Richter. TARGET AUDIENCES: Target audiences for this project are undergraduate and graduate level students, as well as the greater scientific community. Efforts included outreach to K-12 students and teachers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
New knowledge has been generated during the project period relating to the ability of a new promoter element to drive heat-inducible expression in transgenic mosquitoes, and concerning the novel activation of endogenous small interfering RNAs following infection with an RNA virus. Virus-induced small RNAs were found to derive from the entire length of mosquito mRNA coding genes, and crossed exon-exon boundaries indicating a precursor substrate generated following mRNA maturation. Virus-induced small RNAs were found to regulate the expression of several host genes, indicating biological relevance. We are currently pursuing the mode of action concerning the biogenesis of these molecules. Concerning our novel promoter elements, following the publication of this work we have already received numerous requests for plasmid constructs by other research groups, indicating that our work is already making an impact on the field.
Publications
- Adelman, Z.N., Anderson, M.A.E., Liu, M., Zhang, L., and Myles, K.M. (2012) Sindbis virus induces the production of a novel class of endogenous siRNAs in Aedes aegypti mosquitoes Insect Mol. Bio. 21(3), 357-368.
- Carpenetti, T. L. G., Aryan, A., Myles, K. M., and Adelman, Z. N. (2012) Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti. Insect. Mol. Bio. 21(1), 97-106.
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