Progress 08/01/11 to 01/31/14
Outputs
Target Audience: Target Audience for duration of project: Year 1 (August 2011- July 2012) 1) Since the beginning of my fellowship I mentored three undergraduates (one female), two from the University of Connecticut (Adam Hejmowski, Daniel Phillips) and one from Yale (Lara Zipper). I also mentored an underrepresented graduate student in science (Gabriel Lozano). All of the undergraduates/graduate students worked with me on my AFRI sponsored research. I taught undergraduates a range of skills from extracting insect and symbiont DNA and RNA, conducting conventional and quantitative PCR, cloning PCR gene fragments, conducting fitness trials on aphids, rearing aphids, growing plants for insect cultures, and dissecting symbionts out of aphids. I taught the graduate student how to filter bacterial cells from insect cells, image them using vital dyes with microscopy, and how to use cutting edge cell sorting techniques. 2) On May 29-June 2, 2012 I attended the i5k insect genomics workshop and presented my AFRI research at the subsequent Arthropod Genomics conference in Kansas City, MO Year 2 (August 2012- July 2013) 3) I attended and presented my AFRI research at the AFRI meeting in Washington D.C. on August 15-17th, 2012 4) From September 24th -28th 2012, I co-taught and organized a 5 day genomics workshop with my colleague Patrick Degnan at University of South Bohemia in Cesky Budejoice in the Czech Republic. The workshop was paid for and sponsored by the University of South Bohemia. We taught over forty doctoral and postdoctoral students in this intense computational and conceptual genomic workshop. 5) On October 22-26, 2012 I was invited to present my AFRI research at ASM's (American Society of Microbiology) beneficial microbe meeting 6) Continued to mentor a female undergraduate on aphid nutrition and symbionts; I taught her how to extract DNA/RNA, conduct PCR, and gel electrophoresis on insect/microbe samples (Lara Zipper from Yale University) 7) Presented my AFRI research at the 2nd annual CT symbiosis symposium at University of Connecticut in Storrs, CT on March 22nd, 2013 8) Wrote an accepted proposal for a symposium (based on results and networking from my AFRI research) entitled “Effects of microbes on insect-plant interactions” for the annual Entomological Society of America conference for November 2013 in Austin, Texas (this symposium was recently nominated) Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1) (Experimentation in the wet and dry lab) I successfully conducting tedious aphid-plant manipulation experiments and genomic data analysis for entire project duration (years 1 and 2) 2) Fall 2011, Attended 6 workshops at Yale for Research Ethics training 3) On September 10-14th, 2012 I attended a genomics workshop in Baltimore sponsored by the University of Maryland, and learned a variety of new computational techniques 4) Attended an intensive 9 week Yale scientific writing course from January 11 – March 22, 2013 5) Wrote an accepted proposal for a symposium (based on results and networking from my AFRI research) entitled “Effects of microbes on insect-plant interactions” for the annual Entomological Society of America conference for November 2013 in Austin, Texas (this symposium was recently nominated) 6) Attended a seminar on Behind the Scenes at NSF, DOE, DOD and Other Funding Agencies: An Insiders' Perspective on Grant Review at Yale on April 4th 2013 How have the results been disseminated to communities of interest? 1) On May 29-June 2, 2012 I attended the i5k insect genomics workshop and presented my AFRI research at the subsequent Arthropod Genomics conference in Kansas City, MO 2) I attended and presented my AFRI research at the AFRI meeting in Washington D.C. on August 15-17th, 2012 3) From September 24th -28th 2012, I co-taught and organized a 5 day genomics workshop in Cesky Budejoice in the Czech Republic. 4) On October 22-26, 2012 I was invited to present my AFRI research at ASM's (American Society of Microbiology) beneficial microbe meeting 5) Presented my AFRI research at the 2nd annual CT symbiosis symposium at University of Connecticut in Storrs, CT on March 22nd, 2013 What do you plan to do during the next reporting period to accomplish the goals? This is a final report however I am currently applying for an extension: -Funding for this project will end on July 31st, 2013. A 6-month extension is proposed to complete the project’s objectives. Currently samples resulting from over one year of experiments are in the final stages before sample submission for microscopy (TEM) and proteomics analysis. Three manuscripts on the project’s research are currently being prepared, however they will not be finalized and submitted until after data submission, output, and analysis are complete. Consequently, an extension of the fellowship deadline is needed to successfully complete data submission and analysis for TEM and proteomics analyses, to maintain insect lines, and to help defray the cost of publishing (especially in open access journals). c. An estimate of funds expected to remain unobligated on the scheduled expiration date; -Estimated funding expected to remain after July 31st, 2013 is ~$7,000. d. A projected timetable to complete the portion(s) of the project for which the extension is being requested; and 8-31-2013 9-31-2013 10-31-2013 11-31-2013 12-31-2013 1-31-2014 -Proteomics final data submission - Maintain insect lines -Analyze data and write up manuscripts -TEM data submission -Analyze data and write up manuscripts - Maintain insect lines Analyze data and write up manuscripts -Submit last round of samples for TEM - Maintain insect lines Analyze data and write up manuscripts - manuscript publication - Maintain insect lines Analyze data and write up manuscripts - manuscript publication - Maintain insect lines Write up manuscripts - manuscript publication - Maintain insect lines
Impacts
What was accomplished under these goals?
I.) To elucidate gene regulation and evolution of the obligate aphid nutritional symbiont Buchnera: I successfully isolated, sequenced, and analyzed Buchnera small RNAs using directional RNAseq from the obligate symbiont Buchnera aphidicola from 4 divergent aphid species that are known as agricultural pests Acyrthosiphon pisum (strains LSR1 and 5A), Acyrthosiphon kondoi (strain Ak), Schizaphis graminum (strain Sg), and Uroleucon ambrosiae (strain UA002, referred to as Ua). a) My results reveal that tRNA regulation and evolution in divergent Buchnera species share similar evolutionary patterns in the loss of key regulatory sequences and pathways involved in tRNA maturation and activation. These patterns reveal the specific mechanisms that reduce the fidelity of Buchnera protein translation (i.e. resulting in less stable and mutated proteins). In addition, novel 3’ tRNA compensatory processes were discovered in this very project and contribute greatly to the fundamental science of tRNAs and gene regulation in microbes with reduced genomes (which are predominately found as nutritional symbionts in insects; especially herbivore pests) (Hansen et al. NAR. 2012) b) In a second component of this project I successfully identified conserved small RNAs in divergent Buchnera that may be responsible for bacterial gene regulation. 60 Buchnera coding sequences that were cis to identified conserved small RNAS were screened for differential RNA expression among three different Buchnera and aphid developmental stages. A proteomic study for two of these key Buchnera lifestages was recently conducted to analyze gene regulation at the protein level in addition to the RNA level. Our results uncover a novel mechanism of gene regulation via small RNAs in these reduced bacterial genomes. This is the first study ever to release these findings. A paper is currently submitted on these exciting new results (Hansen et al. submitted). II.) To elucidate how integrated aphid metabolic genes and its nutritional symbiont interact on different host plants that vary in nutritional content for the aphid (using 1 aphid species): I successfully conducted plant manipulation experiments on alfalfa and fava bean with Acyrthosiphon pisum (strain LSR1) (a less and more nutrition host plant for the aphid respectively). RNAseq was conducted successfully on aphid RNA for 3 aphid treatments on fava and three aphid treatments on alfalfa (metabolic differences were found in aphids feeding on different host plant types that were related to the integrated metabolism). Amino acid concentrations were successfully measured in aphids (e.g. bacteriocytes) for each treatment (significant differences in free amino acids were identified between host plant treatments). Aphid fitness was measured between aphid lines feeding on different host plants (lower in alfalfa feeding aphids versus fava). Also, obligate nutritional symbiont titer (Buchnera) and bacteriocyte count (specialized aphid cells Buchnera resides in) was measured between aphids (not significantly different between treatments). Bacteriocyte images between different host plant treatments using TEM are currently in progress to further investigate the mechanisms behind metabolic differences uncovered by free amino acid analysis and RNAseq. Overall these results demonstrate that the insect metabolically regulates its nutritional symbiont to obtain enough nutrients (essential amino acids) when feeding on a lower nutrient host plant (e.g. alfalfa compared to fava bean) using RNAseq, proteomics, and free amino acid analysis techniques. These exciting new results are currently being written up for two publications (TEM in progress), and now pest development strategies can begin to be developed that target these insect and microbe specific genetic mechanisms. One paper was just accepted on this research (Hansen and Moran. Molecular Ecology. In press) and 2 more manuscripts are currently in progress (Hansen et al. and Hansen) All these data from (I. and II.) are invaluable resources for the public for the annotation of the model herbivore pest insect-microbe system; aphid and Buchnera genomes (e.g. Bacterial and insect RNAseq (17 samples) and proteomics (2 samples)), will be/was released to NCBI when published/submitted. In addition results under both I. and II contribute extensively to understanding how the insect and microbe collaborate together to regulate the insect and microbe’s integrated metabolism. Novel regulatory mechanisms in both the symbiont and insect were discovered in this AFRI project. These mechanisms now identified are most likely universal in many pest herbivore microbe systems (Hansen and Moran. Molecular Ecology. In press), in turn they can be targeted with pest development strategies (e.g. transgenic crops).
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2012
Citation:
Hansen, A.K. and N.A. Moran. 2012. Altered tRNA characteristics and 3' maturation in bacterial symbionts with reduced genomes. Nucleic Acids Research. 2012 Jun 11 [Epub ahead of print].
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2013
Citation:
Hansen, A.K. and N. A. Moran. In press. The impact of microbial symbionts on host plant utilization by herbivorous insects. Molecular Ecology.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Hansen, A.K., Degnan, P., and N. A. Moran. Conserved small RNA expression in divergent reduced genomes from an insect nutritional symbiont.
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Progress 08/01/11 to 07/31/12
Outputs
OUTPUTS: Outputs from this research range from a variety of activities and events that enhanced my professional development in teaching, mentoring, networking, and disseminating of results generated from this AFRI sponsored research. Report outputs completed during the reporting period that contribute to the goals and objectives of the project include mentoring undergraduates, organizing and teaching a workshop on genomics, attending two workshops on genomics, and presenting my AFRI research at three meetings. Since the beginning of my fellowship I mentored three undergraduates, two from the University of Connecticut and one from Yale. All of the undergraduates worked with me on my AFRI sponsored research. I taught undergraduates a range of skills from extracting insect and symbiont DNA and RNA, conducting conventional and quantitative PCR, cloning PCR gene fragments, conducting fitness trials on aphids, rearing aphids, growing plants for insect cultures, and dissecting symbionts out of aphids. From September 24th -28th 2012, I co-taught and organized a 5 day genomics workshop with my colleague Patrick Degnan at University of South Bohemia in Cesky Budejoice in the Czech Republic. The workshop was paid for and sponsored by the University of South Bohemia. We taught over forty doctoral and postdoctoral students in this intense computational and conceptual genomic workshop. From this valuable experience I learned a lot about organizing and teaching a genomic workshop on bacteria and eukaryotes. On May 29-June 2, 2012 I attended the i5k insect genomics workshop and presented my AFRI research at the subsequent Arthropod Genomics conference in Kansas City, MO. I also attended and presented at the AFRI meeting in Washington D.C. on August 15-17th, 2012. On September 10-14th, 2012 I attended a genomics workshop in Baltimore sponsored by the University of Maryland, and learned a variety of new computational techniques. Recently, on October 22-26, 2012 I was invited to speak at ASM's beneficial microbe meeting. All of these activities and events contributed significantly to my professional development in mentoring, teaching, presenting, and networking. Additionally I interviewed and received offers for a few assistant professor positions during my AFRI fellowship. I hope to start a tenure track assistant professor position in Entomology starting in August 2013, continuing research that I started during my AFRI post-doc. PARTICIPANTS: Allison K. Hansen is the PI on this project and she conducted all the research and wrote all the manuscripts for this project. She also mentors and trains undergraduates and taught a genomic workshop. Activities such as workshops and conferences (detailed in the previous boxes) provided professional development for the PI. In addition the PI attended Yale sponsored workshops for Ethics training and Job interview prep. Yale University is where the P.I. is conducting a post-doc in Nancy Moran's lab. This academic institution supplements financial support and supplies, facilities, and equipment for my AFRI fellowship research. I trained three undergraduate students in molecular genetic and entomology skills. Two are from University of Connecticut (Adam Hejmowski, Daniel Phillips), and one is from Yale University (Lara Zipper). In addition I mentored a rotation graduate student at Yale on my Afri project (Gabriel Lozano). TARGET AUDIENCES: One of the undergraduates I mentor includes an individual that is unrepresented in science especially in entomology (women, Lara Zipper). The genomics workshop I taught included international students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Broadly the overall objective of my AFRI research is to further understand how the integrated metabolism between pest insects and their obligate nutritional symbionts function. My current research is unraveling the mystery of how some of these obligate unculturable tiny genome symbionts regulate and achieve core processes such as translation and gene regulation that is required for their survival, and ultimately the survival of their pest insect host. Fundamental advances in bacterial genetics were recently made from my AFRI research that uncover novel mechanisms of translation that most likely can be generalized to other obligate insect symbionts required for insect host survival (Hansen and Moran 2012, NAR). The next step is for this fundamental knowledge to be utilized in pest management. The genetic oddities of these tiny symbionts in translation present great specific targets for pest control since these systems are unlike other free-living bacteria or Eukaryotes, preventing non-target impacts. Additionally over 70 novel small RNAs were found among four divergent Buchnera Taxa (obligate symbiont of divergent aphids) spanning over 70 MY in divergence. My data is currently in prep and suggests that some of these conserved small RNAs play a regulatory role for core information transfer processes of Buchnera, particularly between different aphid and Buchnera life stages (manuscript in prep). Similar to above small RNAs tend to be lineage specific, and therefore aphid specific pest control strategies can be developed potentially through the generation of antisense RNAs from transgenic crops. These antisense RNAs would not target any Eukaryotes or bacteria other than the ones aphids harbor for nutritional enrichment. Aphid and Buchnera transcriptomic data is currently being analyzed to examine how conserved insect-microbe integrated metabolism responses are when aphids feed on different agricultural crops. Experiments and dissections took over several months to perform and Illumina RNA-seq of 12 different samples took over 1 month to prepare libraries and sequence. All data appears to be high quality and experiments luckily were successfully executed. The impact of this specific project will be realized very soon.
Publications
- Hansen, A.K. and N.A. Moran. 2012. Altered tRNA characteristics and 3' maturation in bacterial symbionts with reduced genomes. Nucleic Acids Research. 2012 Jun 11 [Epub ahead of print].
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