Source: PENNSYLVANIA STATE UNIVERSITY submitted to
GUT BACTERIA MEDIATE THE PERCEPTION OF CATERPILLARS BY PLANTS AND PARASITOIDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012490
Grant No.
2017-67013-26596
Project No.
PENW-2016-09298
Proposal No.
2016-09298
Multistate No.
(N/A)
Program Code
A1112
Project Start Date
Jun 1, 2017
Project End Date
May 31, 2022
Grant Year
2017
Project Director
Felton, G. W.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Entomology
Non Technical Summary
Chewing insect herbivores cause massive damage during feeding bycrushing plant tissues with their mandibles, thus releasing a vast array of cues (such as salivry proteins) that may be perceived by the plant, which then mobilizes plant defenses. Among those cues are bacteria that are deposited during feeding by some herbivores such as beetles, which suppress plant defenses. We have new,compelling evidence from field-collected caterpillars that their gut bacteria play a critical role in suppressing induced plant defenses in response to herbivory, but the mechanisms appear to differ from other insects in that the bacteria affect the composition of salivary cues. We will test whether gut bacteria mediate the perception of caterpillars by their host plants and their naturalenemies. We will also identify specific bacteria responsible for any changes in perception.We will use two field-collected species: Helicoverpa zea and Spodoptera frugiperda, feeding on tomato and maize, and the parasitoid Cotesia marginiventris to test the hypothesis. These insects are each responsible for more than a billion dollars per year in control costs and damage. Toamto and maize are two of the most important crops grown worldwide.Completing these objectives will uncover the overlooked role of gut microbes in mediating plant-herbivore interaction
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2111510113050%
2063110106050%
Knowledge Area
206 - Basic Plant Biology; 211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1510 - Corn; 3110 - Insects;

Field Of Science
1130 - Entomology and acarology; 1060 - Biology (whole systems);
Goals / Objectives
An important strategy used by herbivores to suppress plant defenses is to co-opt their symbiotic microbes to suppress plant defenses. Microbes can provide nutrients to the host, aid in detoxification of plant toxins, regulate host immunity, and provide digestive access to plant nutrients, but recent work shows they may also manipulate plant defense signaling pathways. We reported that the microbe-rich regurgitant of the Colorado potato beetle and the false potato beetle suppress JA regulated defense transcripts (e.g., proteinase inhibitors, polyphenol oxidase (PPO) in tomato. Larvae exploit bacteria in their regurgitantto suppress these defenses, thus deceiving plants into incorrectly perceiving the threat as microbial. Flagellin associated with the bacteria is perceived as a MAMP (microbial-associated molecular pattern) and results in suppression of the JA signaling pathway. Consequently, herbivory by chewing insects encompasses the most complex set of cues that plants must process--the integration of DAMPs, HAMPs, effectors and MAMPs. Yet the role of gut bacteria of other chewing insects such as caterpillars has been sorely neglected in studies of herbivore-induced plant defenses.Virtually all studies to date focusing on HAMPs or effectors in caterpillars have used lab colonies. This is significant for several reasons: lab colonies of many caterpillars have significantly lower diversity and abundance of gut bacteria than field collected larvae (28, 29); artificial diets (containing antibiotics) reduce gut bacterial diversity; and the implications of the exposure of caterpillars to a more complex bacterial community when feeding on host plantsare largely not known. The specific aims/goals of our project are to:Determine how caterpillar gut bacteria mediate the induction of plant defensesIdentify specific bacteria mediating perception of caterpillarsDetermine if caterpillar gut bacteria affect host finding by parasitoidsCompletion of our goalswill provide novel and compelling evidence that the third trophic level (i.e., bacteria) plays an essential role in mediating plant and parasitoid perception of herbivory.
Project Methods
H. zea and S. frugiperda caterpillars will becollected from several geographical locations to ensure we capture a range of their potential microbial diversity. We will isolate bacteria from their guts and oral secretions and use culture dependent and independent methods to identify their resident bacteria. We will then re-introduce culturable bacteria to lab reared caterpilalrs to determine their effect on tha ability of caterpillars to elicit defenses in both tomato and maize. Furthermore, we will use antibiotic treatments of field-collected larvae to assess whether bateria play a role in the ability of caterpillars to elicit plant defenses.We will conduct a thorough survey to determine if the salvary glands of field-collected larvaeharbor any bacteria. The role of gut or salivary gland bacteria in modifying the composition of salivary proteins will be assessed using proteomic methods.In the case of the fall armywormwhere we observed that deposited bacteria might directly suppress defenses, we will determine the active component(s) for suppression. Several bacterial MAMPS (i.e., lipopolysaccharide or LPS from gram negative bacteria, flagellin, and bacterial elongation factor-Tu (EF-Tu) have been well characterized and could be involved in suppression. Bacteria that suppress plant defenses will be subcultured to obtain pure cultures; single colony isolates will then be grown in liquid media and cells will be collected by centrifugation. Culture supernatants will be filtered through a 0.20 µm filter and proteins will be concentrated with Amicon filters to test proteins of variable molecular weight ranges. Active fractions will be subjected to shotgun proteomicsto identify active proteins. To determine if caterpillar gut bacteria affect host-location by parasitoids we will use the generalist solitary larval endoparasitoid Cotesia marginventris (Braconidae), its host fall armyworm, and corn and tomato plants. Third instar larvae will be used for these experiments, as C. marginiventris prefers to parasitize young caterpillars. We will determine if parasitoids demosntrate a behavioral preference for axenic caterpilalrs vs. those possessing gut bacteria. Axenic or bacteria-free caterpillars will be created by surface-sterilization of egg masses in 5% sodium hypochlorite for 2 min; larvae will then be reared autoclaved artificial diet. We have demonstrated that this method produces apparently healthy and bacteria-free caterpillars, which has been confirmed by culturing caterpillar regurgitant and16S sequencing of caterpillar hemolymph, salivary glands, midgut, Malpighian tubules and fat body.

Progress 06/01/17 to 05/31/22

Outputs
Target Audience:Our targeted audiences have been plant biologists, microbiologists, and entomologists. The results of our project have beenpresented at several professional meetings focusing on the Phytobiome, Chemical Ecology, Plant Science, and Entomology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have worked on the project. Jie Wang completed his Ph.D. in China and has now begun a faculty position at Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou, China. Asher Jones completed her Ph.D. and obtained a position as a science writer for the Voice of America. Charles Mason (postdoctoral) obtained a permanent position as a staff scientist with USDA-ARS, Hilo, Hawaii. How have the results been disseminated to communities of interest?Results from the project have been presented at the Entomological Society of America (Vancouver, 2019),Phytobiome Conference (Montpellier, France2019) and at the Gordon Conference in Plant-Herbivore Interactions (Ventura CA 2019). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We conducted a series of assays to determine whether infection with E. ludwigii affects H. zea larval growth, immunity, and salivary responses and thus influences induced defenses of maize to herbivory. This work is now published in the Journal of Chemical Ecology.Inoculating lab-reared caterpillars with E. ludwigii, did not significantly affect the growth of caterpillars, but two immunity-related genesglucose oxidase (GOX) and lysozyme (LYZ) were more highly expressed in both salivary glands and midguts compared withMgCl2 solution-treated caterpillars. Oral elicitors were evaluated for their role in triggering maize-specific defense responses.Our results show that saliva and its main component protein glucose oxidase (GOX) from E. ludwigii-inoculated caterpillars played a role in inducing maize anti-herbivore responses. These findings provide a novel concept that introducing bacteria to an herbivore may be an important approach to pest control through alteration of insect immune responses and thus indirect induction of plant resistance. We have conducted a deeper analysis of gut bacteria from field populations oflarvae collected on maize.We used high-throughput sequencing to determine factors that influence gut microbiomes of field-collected fall armyworm (Spodoptera frugiperda) and corn earworm (Helicoverpa zea). Fall armyworm midgut bacterial communities differed from those of corn earworm collected from the same host plant species at the same site. However, corn earworm bacterial communities differed between collection sites. Subsequent experiments using fall armyworm evaluating the influence of egg source and diet indicated that that host plant had a greater impact on gut communities. We also observed differences between regurgitant (foregut) and midgut bacterial communities of the same insect host, suggesting differential colonization. Our findings indicate that host plant is a major driver shaping gut microbiota, but differences in insect physiology, gut region, and local factors can also contribute to variation in microbiomes. Additional studies are needed to assess the mechanisms that affect variation in insect microbiomes, as well as the ecological implications of this variability in caterpillars. As a result of this analysis, we have identified additional bacteria that we have now cultured and determined their effect on host physiology, salivary elicitors, etc. Additionally, we fund that parasitoids exert a profound impact on gut bacterial communities which, in turn affected the composition of salivary effectors in their caterpillar hosts.We used the fall armyworm (FAW), Spodoptera frugiperda, and the braconid parasitoid Cotesia marginiventris to evaluate impacts of parasitism on the gut microbiome of FAW larvae, and respective maize plant defense responses. The level of reactive oxygen species and the microbial community in larval gut underwent significant changes in response to parasitism, leading to a significant reduction of Enterococcus, while elevating the relative abundance of Pseudomonas. FAW with parasitism had lower glucose oxidase (GOX) activity in salivary glands and triggered lower defense responses in maize plants. These changes corresponded to effects on plants, as Pseudomonas inoculated larvae had lower activity of salivary GOX and triggered lower defense responses in maize plants. Our results demonstrated that parasitism had cascading effects on microbial associates across trophic levels and also highlighted that insect gut bacteria may contribute to complex interrelationships among parasitoids, herbivores, and plants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jones, A. G., Mason, C. J., Felton, G. W., & Hoover, K. (2019). Host plant and population source drive diversity of microbial gut communities in two polyphagous insects. Scientific reports, 9(1), 2792.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Mason, C. J., Jones, A. G., & Felton, G. W. (2019). Co-option of microbial associates by insects and their impact on plantfolivore interactions. Plant, cell & environment, 42(3), 1078-1086.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, J., Yang, M., Song, Y., Acevedo, F. E., Hoover, K., Zeng, R., & Felton, G. W. (2018). Gut-Associated Bacteria of Helicoverpa zea Indirectly Trigger Plant Defenses in Maize. Journal of chemical ecology, 44(7-8), 690-699.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Mason, C. J., St. Clair, A., Peiffer, M., Gomez, E., Jones, A. G., Felton, G. W., & Hoover, K. (2020). Diet influences proliferation and stability of gut bacterial populations in herbivorous lepidopteran larvae. PloS one, 15(3), e0229848.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mason, C. J., Peiffer, M., Chen, B., Hoover, K., & Felton, G. W. (2022). Opposing Growth Responses of Lepidopteran Larvae to the Establishment of Gut Microbiota. Microbiology spectrum, 10(4), e01941-22.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mason, C. J., Peiffer, M., St Clair, A., Hoover, K., & Felton, G. W. (2022). Concerted impacts of antiherbivore defenses and opportunistic Serratia pathogens on the fall armyworm (Spodoptera frugiperda). Oecologia, 198(1), 167-178.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mason, C. J., Peiffer, M., Felton, G. W., & Hoover, K. (2022). Host-Specific larval lepidopteran mortality to pathogenic Serratia mediated by poor diet. Journal of invertebrate pathology, 194, 107818.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sugio, A., Felton, G. W., Giron, D., & Kaloshian, I. (2020). Plant-Arthropod Interactions: Effectors and Elicitors of Arthropods and Their Associated Microbes. Frontiers in Plant Science, 11, 610160.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang, J., Mason, C. J., Ju, X., Xue, R., Tong, L., Peiffer, M., ... & Felton, G. W. (2021). Parasitoid causes cascading effects on plant-induced defenses mediated through the gut bacteria of host caterpillars. Frontiers in microbiology, 12.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mason, C. J., Hoover, K., & Felton, G. W. (2021). Effects of maize (Zea mays) genotypes and microbial sources in shaping fall armyworm (Spodoptera frugiperda) gut bacterial communities. Scientific reports, 11(1), 1-10.
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Chen, B., Mason, C. J., Peiffer, M., Zhang, D., Shao, Y., & Felton, G. W. (2022). Enterococcal symbionts of caterpillars facilitate the utilization of a suboptimal diet. Journal of Insect Physiology, 138, 104369.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chen, X., Peiffer, M., Tan, C. W., & Felton, G. W. (2020). Fungi from the black cutworm Agrotis ipsilon oral secretions mediate plantinsect interactions. Arthropod-Plant Interactions, 14(4), 423-432.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Pan, Q., Shikano, I., Felton, G. W., Liu, T. X., & Hoover, K. (2021). Host permissiveness to baculovirus influences time?dependent immune responses and fitness costs. Insect science, 28(1), 103-114.


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Scientific community including entomologists, plant scientists, and microbiologists. Changes/Problems: Nothing Reported 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 major accomplishment during this period was the discovery that parasitoid regulateinsect-plant-microbiome interactions which result in alteration of the oral secretions of the caterpillar hosts.In this study, we used the fall armyworm (FAW), Spodoptera frugiperda, and the braconid parasitoid Cotesia marginiventris to evaluate impacts of parasitism on the gut microbiome of FAW larvae, and respective maize plant defense responses. The level of reactive oxygen species and the microbial community in larval gut underwent significant changes in response to parasitism, leading to a significant reduction of Enterococcus, while elevating the relative abundance of Pseudomonas. FAW with parasitism had lower glucose oxidase (GOX) activity in salivary glands and triggered lower defense responses in maize plants. These changes corresponded to effects on plants, as Pseudomonas inoculated larvae had lower activity of salivary GOX and triggered lower defense responses in maize plants. Our results demonstrated that parasitism had cascading effects on microbial associates across trophic levels and also highlighted that insect gut bacteria may contribute to complex interrelationships among parasitoids, herbivores, and plants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang J, Mason CJ, Ju X, Xue R, Tong L, Peiffer M, Song Y, Zeng R, Felton GW. Parasitoid Causes Cascading Effects on Plant-Induced Defenses Mediated Through the Gut Bacteria of Host Caterpillars. Front Microbiol. 2021 Sep 6;12:708990. doi: 10.3389/fmicb.2021.708990. PMID: 34552570; PMCID: PMC8452159.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mason, C. J., Hoover, K., & Felton, G. W. (2021). Effects of maize (Zea mays) genotypes and microbial sources in shaping fall armyworm (Spodoptera frugiperda) gut bacterial communities. Scientific reports, 11(1), 1-10.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang, Jie, Charles J. Mason, Xueyang Ju, Rongrong Xue, Lu Tong, Michelle Peiffer, Yuanyuan Song, Rensen Zeng, and Gary W. Felton. "Parasitoid causes cascading effects on plant-induced defenses mediated through the gut bacteria of host caterpillars." Frontiers in microbiology 12 (2021).


Progress 06/01/19 to 05/31/20

Outputs
Target Audience:Our targeted audiences have been plant biologists, microbiologists, and entomologists. The results of our project have been presented at several professional meetings including the Entomological Society of America, Midwest Ecology and Evolution Conference, InternationPhytobiome Meeting (Venice), and International Society of Chemical Ecology (Atlanta). Changes/Problems:The major issue has been the closure of the campus resulting in a near stoppage in research progress this spring. What opportunities for training and professional development has the project provided?Asher Jones has worked on the project and will defend her Ph.D. thesis at the end of May 2020. She has already accepted two internships in Washington DC working on a career in science communication. Postdoctoral scholar Charlie Mason has begun working on the project this Spring. Dr. Mason had a interview for a tenure track faculty position scheduled in April but it was cancelled until further notice due to COVID-19. How have the results been disseminated to communities of interest?Yes. Results have been presented at the Entomological Society of America (2019; St. Louis), International Society of Chemical Ecology (2019; Atlanta), Internatiomal Phytobiome Conference (2019; Vienna) and Midwest Ecology and Evolution Conference (2020, Macomb, Il). What do you plan to do during the next reporting period to accomplish the goals?Progress came to a near halt this spring due to the closure of the campus. When we are able to resume research, we will continue analyzing the effect of the glucose oxidase (GOX) mutation on the maintenance of gut bacteria inHelicoverpa zea. This will allow us to provide more definitive evidence for the role of bacteria in mediating GOX salivary expression and the concomitant effects on the induction of plant defenses. We will also use the GOX mutant lines to determine the role of GOX in mediating perception by parasitoids (Goal 3). In addition, based upon our preliminary data as it appears the GOX regulates at least the quantity of bacteria in the caterpillar's gut, we willconduct a culture-independent 16-S sequencing project to assess the impact of GOX on the bacterial community ofH. zea.

Impacts
What was accomplished under these goals? Most of our accomplishments have focused on goals 1 and 2. We were able to produce axenic caterpillars which has been a great aid in determining the role of gut bacteria in mediating plant induced responses.In the course of conducting experiments to reintroduce bacterial isolates into the axenic caterpillars, we observed that certain isolates great amplified the response of the insect to certain plant defenses in maize. Wetested how particular members present in the gut community influence interactions with plant defenses that can alter PM permeability. We observed interactions between gut bacteria with plant resistance. Axenic insects grew more but displayed lower immune-based responses compared with those possessing Enterococcus, Klebsiella, and Enterobacter isolates from field-collected larvae. While gut bacteria reduced performance of larvae fed on plants, none of the isolates produced mortality when injected directly into the hemocoel. Our results strongly suggest that plant physical and chemical defenses not only act directly upon the insect, but also have some interplay with the herbivore's microbiome. Combined direct and indirect, microbe-mediated assaults by maize defenses on the fall armyworm on the insect digestive and immune system reduced growth and elevated mortality in these insects. We are excited that we have used CRISPR-Cas 9 to produce to mutant, knockout lines ofHelicoverpa zeawhich will allow us to provide more definitive evidence for the role of GOX in mediating the responses we have thus far observed.Pre-blastoderm embryos of H. zea were injected with Cas9 protein and gRNAs targeting two sites located on exon 4 of the gox gene (sites 4.1 and 4.2). All individuals that survived to adulthood after injection were sequenced and those with indels at either target site were identified and intercrossed randomly to generate families. We successfully obtained a family that produced viable G1 offspring and from these we obtained a subsequent family (AA22 female and AA02 male) that produced viable G2 offspring with two distinct mutant alleles (gox-M1 and gox-M2) based on their sequence chromatograms. The gox-M1 allele has a 6 bp insertion and 1bp deletion at the 4.1 site and an insertion of 7 bp at the 4.2 site, which led to a premature stop codon in exon 4. The gox-M2 allele has an insertion of 2 bp on the 4.2 site, creating a frame-shift mutation. We intercrossed homozygous individuals for each allele independently and obtained four homozygous lines from parents carrying the gox-M1 allele (M1-33/02, 19/39, 97/85, 6/36) and four homozygous lines carrying the gox-M2 allele (M2-99/21, 67/53, 5/54, 8/38). To screen for GOX activity individuals in G2 caterpillars, a paper-filter-based qualitative enzymatic assays was performed. All individuals with the mutant alleles gox-M1 and gox-M2 had low to almost no measurable GOX activity compared to wildtype individuals. After isolating eight homozygous lines carrying these alleles in the G3 generation, we randomly selected 10 caterpillars from each line, extracted their salivary glands and performed the GOX activity assay and gox mRNA quantification by qPCR. The mRNA expression levels (Chi-squared = 11.95, df = 2, p = 0.003, Kruskal-Wallis test) and GOX activity (Chi-squared = 12.77, df = 2, p = 0.002, Kruskal-Wallis test) were significantly lower for lines carrying the two mutant alleles gox-M1 and gox-M2 compared to wildtype. Our preliminary analyses (completed just prior to the COVID-19 closure) indicate that GOX plays a significant role in mediating the quantity of bacteria found in the oral secretions and gut of the caterpillar. The gox-M1 mutant line possesses nearly 10 fold higher levels of culturable bacteria in their oral secretions. Subsequent bacterial community analyses will take place once we can return to the laboratory.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Mason, C. J., St. Clair, A., Peiffer, M., Gomez, E., Jones, A. G., Felton, G. W., & Hoover, K. (2020). Diet influences proliferation and stability of gut bacterial populations in herbivorous lepidopteran larvae. Plos one, 15(3), e0229848.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chen, X., Peiffer, M., Tan, C. W., & Felton, G. W. (2020). Fungi from the black cutworm Agrotis ipsilon oral secretions mediate plantinsect interactions. Arthropod-Plant Interactions, 1-10.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jones, A. G., Hoover, K., Pearsons, K., Tooker, J. F., & Felton, G. W. (2020). Potential Impacts of Translocation of Neonicotinoid Insecticides to Cotton (Gossypium hirsutum (Malvales: Malvaceae)) Extrafloral Nectar on Parasitoids. Environmental entomology, 49(1), 159-168.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Paudel, S., Lin, P. A., Foolad, M. R., Ali, J. G., Rajotte, E. G., & Felton, G. W. (2019). Induced Plant Defenses Against Herbivory in Cultivated and Wild Tomato. Journal of chemical ecology, 45(8), 693-707.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mason, C. J., Ray, S., Shikano, I., Peiffer, M., Jones, A. G., Luthe, D. S., ... & Felton, G. W. (2019). Plant defenses interact with insect enteric bacteria by initiating a leaky gut syndrome. Proceedings of the National Academy of Sciences, 116(32), 15991-15996.


Progress 06/01/18 to 05/31/19

Outputs
Target Audience:Our targeted audiences have been plant biologists, microbiologists, and entomologists. The results of our project have been presented at several professional meetings focusing on the Phytobiome, Chemical Ecology, Plant Science, and Entomology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have worked on the project. Jie Wang completed his Ph.D. in China and has now begun a faculty position at Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou, China. Asher Jones has worked on the project and passed her comprehensive exam in the Fall. She has submitted two papers on her work related to this project, both of which are now published. How have the results been disseminated to communities of interest?Results from the project have been presented at the Entomological Society of America (Vancouver, 2019), Phytobiome Conference (Montpellier, France 2019) and at the Gordon Conference in Plant-Herbivore Interactions (Ventura CA 2019). What do you plan to do during the next reporting period to accomplish the goals?With the completion of the microbiomes of the caterpilalrs, we have several additional bacteria to examine. These bacteria (Methylobacteria) require different culture media and we have developed the protocols now to test them in their caterpillar hosts. We are completing experiments on the effects of the bacteria on parasitoid behavior.

Impacts
What was accomplished under these goals? We conducted a series of assays to determine whether infection with E. ludwigii affects H. zealarval growth, immunity, and salivary responses and thus influences induced defenses of maize to herbivory. This work is nowpublished in the Journal of Chemical Ecology. Inoculating lab-reared caterpillars with E. ludwigii, did not significantly affect thegrowth of caterpillars, but two immunity-related genes glucose oxidase (GOX) and lysozyme (LYZ) were more highlyexpressed in both salivary glands and midguts compared with MgCl2 solution-treated caterpillars. Oral elicitors wereevaluated for their role in triggering maize-specific defense responses. Our results show that saliva and its main componentprotein glucose oxidase (GOX) from E. ludwigii-inoculated caterpillarsplayed a role in inducing maize anti-herbivore responses. These findings provide a novel concept that introducing bacteria toan herbivore may be an important approach to pest control through alteration of insect immune responses and thus indirectinduction of plant resistance. We have conducted a deeper analysis of gut bacteria from field populations of larvae collected on maize. We used highthroughputsequencing to determine factors that influence gut microbiomes of field-collected fall armyworm (Spodopterafrugiperda) and corn earworm (Helicoverpa zea). Fall armyworm midgut bacterial communities differed from those of cornearworm collected from the same host plant species at the same site. However, corn earworm bacterial communities differedbetween collection sites. Subsequent experiments using fall armyworm evaluating the influence of egg source and dietindicated that that host plant had a greater impact on gut communities. We also observed differences between regurgitant(foregut) and midgut bacterial communities of the same insect host, suggesting differential colonization. Our findings indicatethat host plant is a major driver shaping gut microbiota, but differences in insect physiology, gut region, and local factors canalso contribute to variation in microbiomes. Additional studies are needed to assess the mechanisms that affect variation ininsect microbiomes, as well as the ecological implications of this variability in caterpillars. As a result of this analysis, we ahve identified additional bacteria that we have now cultured and begun to determine theireffect on host physiology, salivary elicitors, etc.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Jones, A. G., Mason, C. J., Felton, G. W., & Hoover, K. (2019). Host plant and population source drive diversity of microbial gut communities in two polyphagous insects. Scientific reports, 9(1), 2792.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Mason, C. J., Jones, A. G., & Felton, G. W. (2019). Co-option of microbial associates by insects and their impact on plantfolivore interactions. Plant, cell & environment, 42(3), 1078-1086.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, J., Yang, M., Song, Y., Acevedo, F. E., Hoover, K., Zeng, R., & Felton, G. W. (2018). Gut-Associated Bacteria of Helicoverpa zea Indirectly Trigger Plant Defenses in Maize. Journal of chemical ecology, 44(7-8), 690-699.


Progress 06/01/17 to 05/31/18

Outputs
Target Audience:Our targeted audiences have been plant biologists, microbiologists, and entomologists. The results of our project have been presented at several professional meetings focusing on the Phytobiome, Chemical Ecology, Plant Science,and Entomology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students have worked on the project. Jie Wang completed his Ph.D. in China and has now begun a faculty position atKey Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou, China. Asher Jones has worked on the project and will be taking her comprehensive exam in the Fall. She is close to completing a project and will be submitting her work for publciation. How have the results been disseminated to communities of interest?Results from the project have been presented at Phytobiome Conferences/Symposia in 2018 at the University of Hawaii and Penn State University. Presentations in 2018 have also been to the Entomology Department, University of California, Davis and at the Second International CRC 973 Symposium "Bridging Ecology and Molecular Biology: Organismic Responses to Recurring Stressin Berlin, Germany. For 2017 invited symposium talks were presented at the Annual Meeting of the International Society of Chemical Ecology (Kyoto, Japan) and the Entomological Society of America (Denver, CO). What do you plan to do during the next reporting period to accomplish the goals?We are comparing the microbiomes of the fall armyworm and corn earworm and plan to submit this work in 2019 for publication. Efforts on Goal 3 continue as we develop improved protocols for parasitoid behavior.

Impacts
What was accomplished under these goals? Most of the completed work has focused on Goals 1 and 2 during the first year. Experimental procedures are beig developed for Goal 3. Progress towards Goal 1& 2: The polyphagous herbivore Helicoverpa zea is a major agricultural pest that harbors certain bacteriain their digestive systems. Enterobacter ludwigii is one of the gut-associated bacteria commonly identified from field-collected caterpillars, and it has been shown to indirectly induce defenses in the dicot plant tomato by triggering the biosynthesis of salivary elicitors, but there are no clear mechanisms to show how gut microbes alter these salivary cues and how a different host plant responds to these inducible elicitors. Here, we conducted a series of assays to determine whether infection with E. ludwigii affects H. zea larval growth, immunity, and salivary responses and thus influences induced defenses of maize to herbivory. Inoculating lab-reared caterpillars with E. ludwigii, did not significantly affect the growth of caterpillars, but two immunity-related genes glucose oxidase (GOX) and lysozyme (LYZ) were more highly expressed in both salivary glands and midguts compared with MgCl2 solution-treated caterpillars. Oral elicitors were evaluated for their role in triggering maize-specific defense responses. Our results show that saliva and its main component protein glucose oxidase (GOX) from E. ludwigii-inoculated caterpillars played a role in inducing maize anti-herbivore responses. These findings provide a novel concept that introducing bacteria to an herbivore may be an important approach to pest control through alteration of insect immune responses and thus indirect induction of plant resistance. Other related accomplishments: In addition to the role of the bacteria in mediating salivary changs in caterpillars, we have conducted experiments focusing on the impact of these bacteria directly on the host plant. The bacterium, Enterobacter ludwigii, was isolated from the regurgitant of field-collected tomato fruitworm, Helicoverpa zea. The bacterium can be secreted by the insect onto tomato seeds during fruit feeding and is also commonly found in the soil. We applied E. ludwigii to germinated tomato seeds and measured tomato plant growth and productivity under controlled greenhouse conditions. Since there are often trade-offs between plant growth and plant defenses, we examined whether the E. ludwigii-mediated faster growth corresponds with weaker anti-herbivore defenses. When E. ludwigii was applied to germinated tomato seeds, the plants exhibited faster root, shoot and hypocotyl growth, and produced more fruits and seeds than untreated control plants. The plants treated with bacteria exhibited the same activity levels of two key enzymes involved in anti-herbivore defenses, polyphenol oxidase and peroxidase, and induced the same levels of mortality and growth inhibition in H. zea larvae as untreated plants. Thus, our results demonstrate that the application of E. ludwigii to seeds can promote tomato plant growth and yield without compromising anti-herbivore defenses.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, J., Yang, M., Song, Y. et al. Gut-Associated Bacteria of Helicoverpa zea Indirectly Trigger Plant Defenses in Maize. J Chem Ecol (2018). https://doi.org/10.1007/s10886-018-0970-0
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: hikano, I., Pan, Q., Hoover, K. et al. Herbivore-Induced Defenses in Tomato Plants Enhance the Lethality of the Entomopathogenic Bacterium, Bacillus thuringiensis var. kurstaki. J Chem Ecol (2018). https://doi.org/10.1007/s10886-018-0987-4
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Qinjian Pan, Ikkei Shikano, Kelli Hoover, Tong-Xian Liu1, Gary W. Felton. Enterobacter ludwigii, isolated from the gut microbiota of Helicoverpa zea, promotes tomato plant growth and yield without compromising anti-herbivore defenses. Arthropod Plant Interactions. (2018). in press.