2016 Fiscal Year Research-status Report
Molecular studies on nutrient regulation of reproduction and immune responses in a tick
| Project/Area Number |
16K08094
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| Research Institution | University of Tsukuba |
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Principal Investigator |
Taylor DeMar 筑波大学, 生命環境系, 教授 (50261772)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | Target of Rapamycin / soft tick / Ornithodoros moubata / nutrient pathway / vitellogenin |
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| Outline of Annual Research Achievements |
Focus of research for the first year of the project was on identification of the Target of Rapamycin (TOR) kinase gene from the soft tick Ornithodoros moubata. Primers were designed based on the analysis of a 2nd generation sequence obtained from O. moubata and RT-PCR carried out on cDNA samples synthesized from RNA extracted from different stages (during feeding; 2,6 and 12 hr after feeding; 1, 2,3,4,5 and 6 days after feeding) of the O. moubata female ticks.Tri-reagent was used for RNA extraction. cDNA was synthesized with a Superscript III kit. A second set of primers was designed based on high homology regions of this gene in other arthropod sequences,a hard tick,a mite,a cockroach and the fruit fly.The second set of degenerate primers designed from the homologous regions revealed the TOR gene is expressed at similar levels in all stages of O. moubata. Cloning techniques were begun using the actin gene to confirm the gene could be cloned into the pGEM-T Easy Vector System, because we were not able to obtain enough cDNA to carry out direct sequencing. Preparations are now underway to prepare plasmids of the TOR kinase gene so the sequence can be confirmed. Afterwhich, we will prepare double stranded RNA to carry out the RNAi knockdown experiments.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
I expected the analysis of a 2nd generation sequence of the tick would provide a better specific sequence fragment that could be used to pick up the TOR kinase gene. Unfortunately, we could not detect the gene, so we then designed degenerate primers based on high homology regions as originally planned and was able to pick up the gene.This has delayed the identification of the sequence slightly. In addition, two students were hired to carry out the experiments and it took time to train them with the proper techniques.
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| Strategy for Future Research Activity |
The first several months of this year will be spent on sequencing the TOR kinase gene we were able to obtain. Double stranded RNA primers can then be determined and used to create an RNAi system that can be used to knock out the TOR gene and then measure the expression levels of the hormonal receptors and immune receptors that regulate these processes in this tick. Parallel experiments will be ran with inhibition of the TOR gene with injection of rapamycin to confirm the TOR kinase is key in the regulation of reproduction and immune responses in this model tick species.
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| Causes of Carryover |
The original plan was to purchase a NanoDrop 2000 C for approximately 2,150,000 yen, but the amount received would not cover such a purchase and allow for carrying out the research plan. Therefore, arrangements were made to use the equipment of another researcher. The research budget could then be used to support research assistants and provide kits and supplies to carry out the experiments. The second year plan requires the purchase of more expensive molecular kits and supplies. Also more time will be necessary for research assistants to complete the experiments.Results will be presented at domestic and international meetings.
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| Expenditure Plan for Carryover Budget |
Kits for molecular techniques: sequencing, real-time PCR, RNA extraction, RT-PCR, cDNA synthesis, cloning, RNAi, 3'& 5'RACE.Supplies for molecular experiments: tips, tubes, pipetters, glassware, etc.These supplies are needed to identify the TOR and insulin genes and examine the effects of knockout of these genes on reproduction and immune responses.
Research assistants (2 to 3) to carry out the research.Funds are needed to report the results at the following meetings.Domestic meetings: Medical Entomology Annual Meeting (Nagasaki), Acarology Annual Meeting (Kagoshima), Applied Entomology Annual Meeting (Kagoshima. International meeting: Ticks and Tick-borne Diseases (Australia).Funds may also be needed to publish paper(s)in reputable journals.
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