Progress 01/15/16 to 01/14/18
Outputs
Target Audience:The target audience of this work includes members of the scientific and agricultural community, undergraduate and graduate students, and the general public. During this project P.D. (Dr. Chong) has successfully presented this research at scientific conferences in the fields of Evolution, Symbioses, and Molecular Evolution. In addition to an oral presentation at the Evolution conference in Austin, TX in 2016, the P.D. has presented posters at the Gordon Research Conference: Animal-microbe symbioses in West Dover, VT (June 2017) and at the annual Society for Molecular Biology and Evolution Conference in Austin, TX (July 2017), where she also organized a symposium on symbiosis and interactions. She will also be participating in another conference where she will present her current research. Dr. Chong has presented two additional seminars on this work hosted by the Biology Department at The University of Akron (March 2017) and the Department of Biology at the University of Hawaii at Manoa (May 2017). The PD has also communicated this research through a recent publication of an article in a scientific journal (Chong and Moran, The ISME Journal 2018) that is currently available to the academic and research community. The P.D. has mentored three undergraduate students from the University of Texas at Austin including one student from a small rural town (C. Cole) and two non-traditional minority students (D. Tran and E. Umanzor). Undergraduate students actively participated in research and gained valuable experiences and developed skills important for professional and research careers including experiences with molecular techniques (e.g. DNA extraction, PCR, gel electrophoresis, DNA sequencing, and microscopy), insect dissection, data collection and analysis, and animal care. The P.D. has also worked to engage the general public by participating in science education and outreach events sponsored by the University of Texas at Austin, including "Explore UT" (March 2017), which brings together local GK-12 students and other members of the community to learn about educational and research opportunities at the University of Texas. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided several training and professional development opportunities for the P.D. (Dr. Chong). Training opportunities include developing skills in scientific and experimental approaches including insect husbandry and maintaining experimental lab cultures, developing qPCR assays and data collection and analysis using these methods, training in microscopy and microinjection techniques, experience generating high-throughput metagenomic sequence data and performing genomics analyses. The P.D. also gained experiences in preparing manuscripts to summarize and communicate the research conducted and successfully published her work in peer-reviewed scientific journals. Dr. Chong also presented her work at three different conferences and three seminars at multiple universities during the project period and was also able to organize a symposium on "Symbiosis and interactions" at the annual Society of Molecular Biology and Evolution (SMBE) conference held in Austin Texas in 2017. She is also currently co-organizing another symposium on "Genomics and evolution of symbiotic interactions" for the 2018 SMBE conference being held in Yokohama, Japan, where she will also present her current research. By participating in these conferences, Dr. Chong had the opportunity to interact with prominent scientists in her field and was able to establish new collaborations. Specifically, the P.D. has collaborated with Dr. Gordon Bennett (currently at the University of California at Merced) on a project studying the role of differential gene expression in obligate bacterial symbionts of the leafhopper pest, Macrosteles quadrilineatus (Cicadellidae), which was recently published (Bennett and Chong, 2017, G3: Genes, Genomes, Genetics). As a broad extension of this work, Dr. Chong and Dr. Moran have also co-authored an extensive annotated bibliography on Endosymbiosis (Chong and Moran, Oxford Bibliographies, under revision). Additionally, the P.D. mentored three undergraduate researchers during this project and provided students with valuable training in insect rearing and experimentation and molecular techniques including DNA extraction, PCR and sequencing, qPCR, and analyzing genomic data. With the support of this project, Dr. Chong has successfully obtained a new position as an Assistant Professor at the University of Hawaii at Manoa in the Department of Biology. In her new position, using the research, communication, and mentoring skills developed, she will continue to pursue projects building on the genomics and evolution of insect-microbe symbioses in her new lab. How have the results been disseminated to communities of interest?Project results have been presented at multiple conferences and seminars encompassing a diverse audience of scientists. Specifically, this research has been presented to evolutionary biologists (annual Evolution conference in Austin, TX; June 2016), molecular biologists (annual Society for Molecular Biology and Evolution (SMBE) in Austin, TX; July 2017), and symbiosis researchers (Gordon Research Conference: Animal-Microbe Symbioses: Host microbe associations at the foundation of the biosphere in West Dover, VT; June 2017). The more current results from this project will be presented at the annual SMBE conference, which will be held in Yokohama, Japan this summer (July 2018). Results have also been presented to broad biology departments at University of Texas at Austin (October 2016), University of Akron (March 2017), and University of Hawaii at Manoa (May 2017). Additionally, results have been published in peer-reviewed scientific journals (Chong and Moran 2016, Proc. Natl. Acad. Sci.; Chong and Moran 2018, ISME Journal). Some of this work is still ongoing and will be presented at scientific conferences and submitted for publication upon completion. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Intimate interactions between bacteria and animals play a critical role in generating and maintaining diversity in all ecosystems. By studying the dynamics of these symbiotic interactions, we gain a deeper understanding of how interactions between different organisms can promote biodiversity as well as how these systems can be directly impacted by changes in biological and environmental factors. Many insects, including aphids, harbor bacterial symbionts that allow them to feed on plant sap by provisioning hosts with essential nutrients lacking in the host diet. The extent to which hosts are able to regulate their symbionts, and the ability of symbionts to provision nutrients, will directly influence the range of hosts plants. Understanding how hosts regulate microbial partners can reveal key factors in maintaining mutually obligate symbiosis, which can allow for better management of pestiferous insects feeding on crop plants. We use a combination of experimental methods, molecular approaches, and high-throughput sequencing to elucidate the relationship between microbial symbionts and their aphid hosts. Ultimately, we show that the hosts play a significant role in regulating their symbionts and that symbiont abundance significantly varies across different lineages, which influence host fitness. Additionally, genetic diversity in microbial symbionts reflect differences in ability to provisions essential nutrients, which further reinforces the need for hosts to regulate symbionts. This variation potentially can lead to shifts in preferred plant hosts and influence the ability of different lineages to be more significant pests. (I) To determine the role of host and symbiont genotypes and their interactions in regulating symbiosis, we generated a large set of genetically distinct clonal lines of pea aphids (n=39) with known relatedness by performing sexual crosses between four divergent parental lines. We founds differences in egg hatching/survivorship of progeny based on parental crosses, suggesting genetic incompatibilities play an important role in limiting the genetic combinations that result in successful progeny. For parental and surviving progeny clones, we estimated symbiont abundance using quantitative PCR (new symbiont assay developed) and measured multiple metrics of host fitness including, development time, weight at maturity, time to reproduction, and reproductive rate. We find variation in symboint abundance and differences in host fitness based on host genotype and find a significant negative correlation between symbiont abundance and host fitness. Specifically, in a controlled lab setting, we show that host genotypes with more symbionts are less fit compared to lineages with fewer symbionts that have higher reproductive rates. Interesting, these results suggest hosts that maintain lower symbiont levels proliferate more quickly and may represent increased potential to become pest species. Through this objective, the PD has gained valuable experiences by designing experiments, performing insect husbandry, conducting new molecular techniques, and preparing a manuscript, which resulted in a publication in a scientific journal (Chong and Moran. PNAS. 2016). Additionally, the progeny clones from the sexual crosses (novel genetic resources) and the molecular resources (qPCR assays) developed will serve as valuable tools for the research community and for future studies. (II) To test whether degenerative evolution of symbiont genomes drives adaptation and diversification, we generated novel genetic clones (above) and conducted symbiont transfers between several lineages. While we were successful in replacing symbionts in hosts that share genetically similar symbionts, repeated attempts to transfer more divergent symbionts failed to produce successful transfers, resulting in either complete mortality or a significant recovery of the nearly depleted native symbiont populations rather than acquisition of genetically divergent symbionts. Our experimental results suggest that the relationships between host and symbiont genotypes are tightly correlated and that hosts may not be able to survive with genetically divergent symbionts, which may be a result of genetic incompatibilities with specific symbionts and that host are unable to maintain and regulate symbionts that are divergent from their native symbionts. Through this objective, the PD has gained valuable experiences with microscopy, microinjection techniques, and genetic screening of experimental lines. We also generated genomic sequences from several aphid hosts and their symbionts and are characterizing the patterns of genome degradation in symbionts across these lineages. Specifically, we used next-generation sequencing (Illumina HIseq 4000) to generate large metagenomic datasets for multiple strains (n=25) of Buchnera and their aphid host. Following sequence assembly, symbiont sequences were identified and extracted, and complete genomes were annotated. The PD is currently conducting comparative genomic analyses and initial results suggest that the degree of genome degradation significantly varies between lineages and that these patterns of mutation and gene loss determine the degree of functionality of symbionts, which would significantly impact hosts ability to feed on different host plants. Deep sequencing of more divergent aphid hosts revealed that one host species lack the ancient symbiont Buchnera, which is usually found in all aphids. Based on genomic analyses, molecular techniques, and microscopy, we identified a novel Bacteroidetes symbiont, which has replaced the ancient symbiont. Compared to related free-living bacteria, the genome of the Bacteroidetes symbiont is more degenerate (reduced genome size and gene loss), though it retains nearly all genes necessary to synthesize essential amino acids. These results suggest the new symbiont has the genomic features of an obligate symbiont and serves as a functional replacement of Buchnera, which was likely unable to provision nutrients and was eventually eliminated. The PD gained additional experience collecting and analyzing large genomic datasets, using new microscopy techniques, and submitting manuscripts for publication (Chong and Moran 2018, ISME Journal). (III) To further investigate how interactions between host and symbiont genotypes impact symbioses, we are examining the role of symbiont regulation under changing environment and dietary regimes. Preliminary data on the impacts of changing environments (increasing temperatures) show different effects on host fitness across lineages that differ in symbiont abundance, where clones with higher symbiont abundance seem to be more robust under exposure to higher temperatures compared to lineages with lower symbiont abundance. This work in ongoing and through her new position at the University of Hawaii at Manoa, Dr. Chong will continue her work on investigating the role of genetic interaction in regulating symbiosis. We will complete experiments to examine how symbiont regulation influences host fitness under different diet treatments. Collectively, these results will help elucidate the role of genetic interactions in driving the regulation of symbiosis and the potential of pestiferous insects to expand their range of plant hosts and colonize new habitats. Additionally, these results will shed light on the impacts of changing environment on the biodiversity and microbial interactions in non-pest insects that are a part of unique ecosystems.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Chong, R.A. and Moran, N.A. (2018) Evolutionary loss and replacement of an obligate endosymbiont in aphids. The ISME Journal. 11:715726
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
A case of evolutionary loss and replacement of an obligate endosymbiont in aphids. Poster presentation at Society of Molecular Evolution Conference 2017 in Austin, TX.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Evolutionary loss and replacement of an obligate endosymbiont in aphids. Poster presentation at Gordon Research Conference: Animal-microbe Symbiosis 2017 in West Dover, VT.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Moran, N.A. and Chong, R.A. (2018) Endosymbiosis. Oxford Bibliographies. [in revision]
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chong, R. A. and Moran, N. A. (2016) Intraspecific genetic variation in hosts affects regulation of obligate heritable symbionts. Proceedings of the National Academy of Sciences USA 113(46): 13114-13119
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Heritability of obligate symbiont titer and link to aphid host fitness. Oral presentation at Evolution Conference 2016 in Austin, TX.
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Progress 01/15/16 to 01/14/17
Outputs
Target Audience:The target audience of this work includes members of the scientific and agricultural community, undergraduate and graduate students, and the general public. During this project period P.D. (Dr. Chong) has successfully presented this research at a scientific conference (oral presentation at the Evolution conference in Austin, TX) and presented a seminar on this work hosted by the Department of Integrative Biology at the University of Texas at Austin (PopBio seminar at UT Austin). The PD has also communicated this research through publication of an article in a scientific journal (Chong and Moran, Proc. Natl. Acad. Sci. USA. 2016) that is currently available to the academic and research community and will be widely available to the public when the article becomes open access. During the project period P.D. has mentored three undergraduate students from the University of Texas at Austin including one student from a small rural town (C. Cole) and two non-traditional minority students (D. Tran and E. Umanzor). These students actively participated in the research project and gained valuable experiences and developed skills important for professional and research careers. Undergraduate research experiences included molecular techniques (e.g. DNA extraction, PCR, gel electrophoresis, DNA sequencing, cloning, and quantitative PCR), insect dissection and microscopy, data collection and analysis, and animal care. The P.D. has also mentored two underrepresented graduate students from the University of Texas at Austin (Ph.D. student S. Zhao and rotation student N. Xerri). Graduate student training included experimental design, insect husbandry, molecular techniques, and bioinformatic skills. The P.D. has also worked to engage the general public by participating in science education and outreach events sponsored by the University of Texas at Austin (Family Day on October 17, 2015 and Explore UT on March 2016). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided numerous training and development opportunities. (1) Animal husbandry and experimental manipulation. The PD has received extensive training in raising and maintaining large numbers of aphid colonies and has learned to perform sexual crosses using aphid clones. Additionally, the PD has performed numerous fitness estimates, which involves measuring development daily, weighing individual aphids using a microbalance, and counting total number of offspring for individual females. (2) Molecular techniques and microscopy. The PD developed additional molecular techniques including quantitative PCR and also learned new skills in dissection, microinjection, and microscopy. (3) Computational skills. The PD has gained skills in statistical analysis and bioinformatics by using high-throughput methods to sequence, assemble, and annotate and analyze complete symbiont genomes. (4) Science communication. The PD participated and presented project research at a scientific conference (oral presentation at the Evolution conference in Austin, TX). (5) The PD has gained experience preparing a manuscript for publication in a scientific journal [Chong and Moran, Proc. Natl. Acad. Sci. USA. 2016] (6) As a USDA postdoctoral fellow, the PD mentored has trained three undergraduate students and two graduate students during this project period including four students from underrepresented groups. The PD also severed as a mentor for the Undergraduate Diversity Program at Evolution 2016. (7) Activities. The PD's proposed symposium topic on symbiosis for SMBE 2017 meeting (Society for Molecular Biology and Evolution annual conference) was selected and the PD is now organizing and selecting speakers for the accepted symposium on symbiosis. How have the results been disseminated to communities of interest?Results from the project have been disseminated through publication, oral presentation, and direct research experiences. The PD has presented project results at a scientific conference (oral presentation at the Evolution conference in Austin, TX on June 17-21, 2016) and presented a seminar on the project at the University of Texas at Austin (PopBio seminar at UT Austin on October 20, 2016). The results from this project have also been presented through publication as a scientific journal article [Chong and Moran, Proc. Natl. Acad. Sci. USA. 2016], which will be available to the public once it is open access in spring 2017. As a USDA postdoctoral fellow, the PD has also disseminated project results by mentoring and training three undergraduate students and two graduate students during this project period. Results from the project have been shared directly with the public at community outreach events hosted by the University of Texas at Austin. What do you plan to do during the next reporting period to accomplish the goals?To accomplish project goals, the PD will perform symbiont transfers, complete genome and transcriptome data collection and analysis, perform environmental stress experiments, and prepare and submit results for publication. To test whether rapid and degenerative evolution of symbiont genomes drives adaptation and diversification in host populations, Buchnera transfers are being performed. New aphid host lines with a replacement of obligate symbiont genotypes have been established, and we are currently working on performing transfers between more divergent host-symbiont lines. In combination with experimental manipulations, high-throughput genotyping will be used to test examine evolution of symbiont genomes. We have collected next-generation sequencing data for a number of different aphid lineages and have started preliminary data analysis while new transfer-genotypes are being generated. Once data collection and analysis is completed project results will be prepared for publication. To determine how interactions between host and symbiont genotypes impact the regulation of symbiosis under changing environments and dietary regimes, novel combinations of pea aphid and Buchnera genotypes have been generated and their fitness performance will be evaluated under different stressed environmental conditions. Experimental manipulations between genotypes are being initiated and testing should be completed by spring 2017. Following experimental manipulations of host environment, genomic and transcriptome sequence data will be collected and analyzed and results will be submitted fall 2017.
Impacts
What was accomplished under these goals?
The major goal of this project is to understand how obligate insect symbioses are functionally regulated and what role symbioses play in the adaptation and diversification of insect hosts using the obligate symbiosis of pea aphids as a model system. (1) To determine the role of host and symbiont genotypes and their interactions in regulating symbiosis, we used experimental crosses to identify to impacts of host genotype by estimating symbiont titer levels, measured as the ratio of symbiont genome copy number and host genome copy number, and by conducting fitness experiments to estimate host development time and fecundity. We show that symbiont titer levels significantly differ significantly differ between sibling aphid lines that harbor the same symbiont genotype. We find significant differences in host fitness between aphid clones and show that this variation in fitness is correlated with symbiont titer, where higher titer levels are correlated with lower host fitness. The work has been published in a scientific journal (Chong and Moran, Proc. Natl. Acad. Sci. USA. 2016). The PD has gained experience by designing the experiment, performing insect (and plant) husbandry, conducting experimental crosses with aphids, learning new molecular laboratory techniques including Quantitative PCR, using bioinformatic tools, and preparing a manuscript for publication. (2) Using a combination of sexual crosses and the Buchnera transfer technique, enabling the replacement of obligate symbiont genotypes without altering host genotype, we are testing whether rapid and degenerative evolution of symbiont genomes drives adaptation and diversification in host populations. We have successfully generated novel genetic combinations of symbiont and host genotypes through sexual crosses and are currently conducting host-symbiont transfers. Parental aphid clones have been sequenced using Illumina MiSeqV3 and symbiont genomes have been assembled and annotated. Accomplishment of these goals provides the PD with training in experimental design and genetics manipulations in an insect symbiont system. In addition in insect husbandry, the PD has gained experience in microscopy and microinjection techniques as well as skills in bioinformatics by using high-throughput methods to sequence, assemble, and annotate complete symbiont genomes. (3) This project involves designing and performing experiments, learning new techniques and skills, publishing research, participating in conferences and seminars, and outreach and mentorship through scientific training of underrepresented minority groups. As a USDA postdoctoral fellow, the PD mentored has trained three undergraduate students and two graduate students during this project period including four students from underrepresented groups.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chong, R. A. and Moran, N. A. (2016) Intraspecific genetic variation in hosts affects regulation of obligate heritable symbionts. Proceedings of the National Academy of Sciences USA 113(46): 13114-13119
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Heritability of obligate symbiont titer and link to aphid host fitness. Oral presentation at Evolution Conference 2016 in Austin, TX.
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