Item No. 1 of 1

INVESTIGATOR: Hansen, A. K.

PERFORMING INSTITUTION:
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA, ILLINOIS 61801

FUSION OF AMINO ACID METABOLISMS BETWEEN PEST APHID SPECIES AND THEIR OBLIGATE BACTERIAL ENDOSYMBIONTS

NON-TECHNICAL SUMMARY: Many plant-feeding insects harbor bacteria that are required (obligate endosymbionts) and/or not required (facultative endosymbionts) for insect survival and reproduction. The key role of obligate endosymbionts to their sap-sucking insect hosts is nutritional. Obligate endosymbionts, such as Buchnera in aphids, provide essential amino acids deficient in the aphid's sap diet for the benefit of their insect host. Some beneficial facultative endosymbionts protect their insect host from natural enemies; natural enemies play an important role in reducing insect herbivore populations. This project attempts to tease apart how an aphid-Buchnera integrated metabolism functions in diverse aphid species and within a species (obj.1), and with and without facultative endosymbionts (obj.2). This research will contribute broadly to scientific knowledge relevant to agriculture, insect ecology and evolution, and symbiosis. All sap-sucking agricultural insect pests (e.g. psyllids, whiteflies, scale insects) possess obligate endosymbionts, analogous to Buchnera, in addition to facultative endosymbionts. The aphid-endosymbiont symbiosis, particularly the symbiosis of pea aphid and its Buchnera, is a model insect-endosymbiont system that is revolutionizing our understanding of endosymbiont evolution, regulation, and functional roles in hosts. Our proposed project will elucidate whether and how pest aphid species with different feeding ecologies interact with their obligate endosymbiont, Buchnera using a functional genomics approach. Additionally, a greater understanding of how mutualistic facultative endosymbionts metabolically interact with Buchnera and their aphid host will be accomplished from this research. Ultimately, this fundamental knowledge is required to understand which insect and bacterial genes are important for insect nutrition and aphid survival.

OBJECTIVES: Long-term goal: My aim is to further understand the functional roles of bacterial endosymbionts in their sap-sucking insect hosts and to elucidate how both partners interact with one another at a molecular level. Proposed project objective:Within and among aphid species for which ingested phloem varies in amount and profile of amino acids, how is the amino acid metabolism of hosts integrated with that of Buchnera, in terms of complementarity within and between pathways and recycling of ammonia.

APPROACH: Within pea aphid species, aphid clones that feed on different host plants ingest a similar profile and total concentration of essential and nonessential amino acids (Sandström and Pettersson 1994). Nevertheless, the quantity of particular amino acids varies dramatically depending on which host plant species the pea aphid feeds on (Sandström and Pettersson 1994). More specifically, aphids ingest a nine fold greater concentration of alanine on alfalfa (Sverre) relative to broad bean (Major). In contrast,pea aphids ingest over two fold more arginine, histidine, tryptophan, glutamine, tyrosine, and phenylalanine when feeding on broad bean versus alfalfa (Sandström and Pettersson 1994). Whether or not host plant dependent ingestion of variable amino acid amounts modifies the integrated amino acid metabolism of pea aphids and Buchnera, is unknown. In turn we dissected out bacteriocytes (specialized insect cells containing Buchnera) in aphid lines that fed on alfalfa compared to fava (N=3) and conducted RNAseq, proteomics, small RNA, and epigenomic analyses on these different host plant treatments to understand the metabolic shift of the shared insect-microbe metabolism between host plant treatments that vary in amino acids. In addition TEM images were taken of bacteriocytes from aphids feeding on different host plant treatments to test if Buchnera cell density changes between hot plant treatments.

PROGRESS: 2013/09 TO 2014/05
Target Audience: -Outreach and scientific presentations to academic and grower audiences: Entomological Society of America- Entomologists and Growers University of Illinois, Champaign-Urbana. Microbiology Department University of Illinois, Champaign-Urbana. Program in Ecology, Evolution, and Conservation Biology University of California, Riverside- Entomologists University of Wisconsin, Madison. Department of Entomology -Mentoring and educational outreach: During this funding period I also trained and mentored 7 students and a technician on this AFRI project. Students and the technician were trained on different aspects of data collection and analysis that resulted from this project. In addition data from this project was incorporated into classrooms to teach undergraduate students in integrative biology about genomics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I trained 7 students and a technician during this current funding period on this AFRI project. A female academic professional Hongmei Li-Byarlay got the opportunity to rear aphids and conduct host-plant diet treatments on aphids. She also recieved experience in preparing an epigenomic library and preparing DNA for BS-sequencing. Hongmei was also given the opportunity to learn basic UNIX and Perl and was trained to analyze some of the preliminary BS-seq data. Currently, a female graduate student Bushra Minhas in informatics has the opportunity to analyze all of the aphid epigenomic diet treatment data this summer, and is learning how to conduct computational genomics in my lab. Five undergraduates also had the opportunity to conduct research on this AFRI project (e.g. diet trials and Buchnera small RNA experiments) during this funding period (Andrew Jang, Thor Hansen, Victoria Wong, Philip Cannizarro, and Yihui Zhu). One undergraduate (Thor Hansen) even received a research award from the School to conduct aphid diet research in my lab this summer, which is directly related to this AFRI project. How have the results been disseminated to communities of interest? Co-organized a major symposium on the general topic of my AFRI research “The Effect of microbes on insect-plant interactions“ for the Entomological Society of America National Meeting. Austin Texas, USA. (2013). Presented my AFRI research to the scientific community at conferences and universities as noted above for "products" Published three peer-reviewed publications as noted above for "products" What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2013/09 TO 2014/05
What was accomplished under these goals? Host control of integrated host-microbe metabolisms may be key for the maintenance of long-term mutualistic symbioses. Insect regulation of the integrated metabolism potentially is adaptive for the insect to respond to variable amino acid availability in plant sap. Thus, we predict that if an aphid feeds on a host plant with low essential amino acid (EAA) concentrations then the insect would compensate by up-regulating key genes involved in the integrated amino acid metabolism. Ultimately, if the insect host can regulate symbiont-provisioned EAA in response to nutritional demands, then these genetic mechanisms can be gained, modified or lost, potentially leading to changes in host plant breadth over evolutionary time (Hansen and Moran 2013). By better understanding the mechanisms underlying insect-symbiont host-plant specialization we can better manage insect crop pests and potentially their host-plant range. Based on our AFRI research we discovered that the nutritional obligate symbiont of aphids (Buchnera) can regulate its own gene expression at the protein level and potentially uses conserved small RNAs in divergent aphid pest species to do this (Hansen and Degnan in press). For the first time our results indicate that the small genome symbiont of sap-feeding insects may actually regulate its own gene expression and it is not just the aphid in control. Interestingly genes related to the biosynthesis of essential amino acids in Buchnera are differentially regulated depending on aphid lifestage (embryo versus adult), and therefore the aphid may need these highly expressed essential amino acids in high quantities during specific lifestages for the aphid or microbe (Hansen and Degnan in press). In addition, using RNAseq and host plant diet treatments we found that when the generalist pea aphid feeds on a host plant with lower EAA concentrations (alfalfa) specific aphid encoded enzymes involved in the integrated metabolism are up-regulated in bacteriocytes over two-fold higher compared to aphids feeding on fava, a host plant with higher EAA concentrations (fava). Based on our RNAseq data (Hansen unpublished data) pea aphids that feed on host plant treatments that vary in EAA (fava versus alfalfa) differentially express several KEGG pathways related to the biosynthesis of aphid amino acids in aphid bacteriocytes (N=3 biological reps). In turn the aphid may be compensating for essential amino acid limitation when feeding on alfalfa. During this funding period we explored how differential mRNA regulation of the integrated metabolism between host-plant treatments may be occurring. Epigenetic mechanisms such as DNA methylation can influence patterns in mRNA gene expression. DNA methylation may be very important in aphid-Buchnera interactions because methylation was recently found to play an important role in mosquito- Wolbachia interactions (Yixin et al, 2013). Moreover our preliminary data from this funding period on whole genome bisulfite sequencing (BS-Seq) on the pea aphid (2 lanes of Hi-seq- 3 biological reps) reveals bacteriocyte specific DNA methylation when aphids feed on fava. For cytosines detected with or without methylation in two or more biological replicates for each tissue treatment (bacteriocyte versus body) (N=3), we observed that methylation sites are shared, and tissue specific in either the bacteriocyte or body. For methylated CpG sites ~60% of methylated Cs are shared in both tissue types and the remainder are tissue specific. Interestingly, the majority of methylated CHG and CHH sites identified are tissue specific either in the body or the bacteriocyte. In turn, tissue specific DNA methylation may result in differential gene regulation or alternative splicing for genes involved in the host’s integrated metabolism in the bacteriocyte. We are currently analyzing data from alfalfa tissue treatments as well (2 lanes of Hi-seq- 3 biological reps) and will investigate if methylated sites that are different between diet treatments correspond with differential expression of our RNAseq data (collected from the previous funding period). These preliminary data are exciting because this is the first time methylation in an obligate insect-microbe symbiosis has been explored. These results will help elucidate the regulatory networks of insect-microbe integrative nutritional metabolisms, and will help out target key genes responsible for increasing host-plant breadth.

PUBLICATIONS (not previously reported): 2013/09 TO 2014/05
1. Type: Journal Articles Status: Published Year Published: 2013 Citation: Hansen, A.K. and N.A. Moran. The impact of microbial symbionts on host plant utilization by herbivorous insects. Molecular Ecology. Article first published online: 16 AUG 2013.
2. Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Hansen, A.K. and P.H. Degnan. Widespread Expression of Conserved Small RNAs in Small Symbiont Genomes. ISME J. In press 2014.
3. Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Casteel, C.L. and A.K. Hansen. In final revision. Evaluating insect-microbiomes at the plant-insect interface. Journal of Chemical Ecology. Invited review.