Source: AUBURN UNIVERSITY submitted to NRP
INVESTIGATING THE VIROMES OF THE INSECT VECTORS OF PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030491
Grant No.
2023-67014-39557
Cumulative Award Amt.
$219,848.00
Proposal No.
2022-08805
Multistate No.
(N/A)
Project Start Date
Apr 1, 2023
Project End Date
Mar 31, 2026
Grant Year
2023
Program Code
[A1112]- Pests and Beneficial Species in Agricultural Production Systems
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
(N/A)
Non Technical Summary
Viruses are valuable members of the biosphere. Viruses are the most abundant source of genetic information in the environment; however, our limited knowledge of their sequences has left us at a disadvantage.They play an important role in the insect vectors of plant pathogens as well. The viruses in insects can be split into multiple categories: insect-specific viruses (ISVs), plant and insect-infecting arboviruses, plant-only infecting viruses, and the microbiome-infecting viruses that interact with the insect endosymbiotic communities.Four categories of viruses in insects have been identified including insect-specific viruses (ISVs), dual plant and insect infecting viruses, plant-only infecting viruses, and insect microbiome viruses. With the diversity of viruses in nature, a variety of relationships between viruses and the endosymbionts can occur in insects. Insects harbor endosymbionts that are critical for their success and can modulate their lifecycle and reproduction. However, once viruses are introduced, the picture becomes more complicated. This project seeks to obtain new viral information regarding three insect species, two thrips species that vector agronomically important viruses and one aphid species which is a cosmopolitan species that has been found to transmit several different agriculturally important viruses including Cotton leafroll dwarf virus and Citrus tristeza virus.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21140301101100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1101 - Virology;
Goals / Objectives
Approximately 76% of plant viruses are transmitted by insect vectors and it is unclear what the presence of insect-specific viruses (ISVs) does to the transmission relationship between plant viruses and insects. In the case of the plant infecting rhabdoviruses and bunyaviruses, it is hypothesized that these viruses were originally insect viruses that gained the ability to infect plants through the acquisition of movement and silencing proteins. This hypothesis was derived from the identification of ISVs of both families. These related ISVs retain enough sequence similarity to be identified through BLAST searches. Due to their similarities, it is also possible that ISVs and these similar dual plant and insect infecting viruses may be able to recombine and reassort to generate new viruses which may become problems in the field as newly emergent plant viruses. Our hypothesis is that the sequencing of F. fusca and A. gossypii will lead to the identification of novel insect viruses and additional plant viruses that can be vectored by these insects and already are or may become a problem in the future. Sequencing of N. variabilis and comparison to the Midwest collection will allow for identification of possible geographic limits to insect viruses in the United States.The proposed project has two objectives:Objective 1: Sequence the viromes of three wild-collected populations of insects, F. fusca, N. variabilis andA. gossypii, to determine the similarities and differences in their viromes in a smaller geographic area (Alabama)Objective 2: Analyze the sequences of viruses in insects that have previously been deposited in Genbank and identify the similarities and differences.Identification of virome sequence data from Genbank: Genbank contains a growing collection of virome data from individual species after analysis such as what we are describing. However, the comparison of these viruses is often limited to a BLAST analysis once the paper is deposited and not revisited when new data becomes available. Earlier virome papers identified viruses that had no known or very little sequence similarity to other viruses. It is necessary to reanalyze the early datasets and the newer datasets with a fresh eye to identify any trends that may have been missed. Comparison of virome sequence data: We propose to pull the sequences that are publicly available in Genbank and rerun the comparison of these viruses to each other. Using the collectioned data from the literature, we will characterize these populations as wild-caught or caged and determine if there are any unidentified trends that should not be ignored.
Project Methods
Sequence the viromes of threewild-collected populations of insects.Wedefinewild-caught as a population of thrips found on the agronomic crop of interest collected from the field in Alabama and not one caged for any length of time. This wild-caught population should have the capacity to both breed in the wild and feed on a variety of different plants. This enables the maximal amount of vertical and horizontal transmission (from parent to child, and from individuals in the same generation), as well as the acquisition of any possible viruses from plant hosts in the wild. Using wild-caught insects instead of cultured, caged insects should increase the variety of viruses due to the free flow of all genetic information both viral and endosymbiotic.Sample selection:From April-June, flights ofF.fuscaare present on peanuts in the fields of Alabama. A population ofno fewer than 100 individuals will be collected live from plants on the fields using aspirators to collect into Eppendorf tubes. Once populations have been identified to beF.fusca,the populations will be stored in 95% ethanol in a-20-degreefreezer until RNA extraction and preparation of samples for sequencing.As part of the 2023 collection ofN. variabilis,a subset of insects will be collected from soybeanfieldsin Auburn (primarily the Old Rotation and theCullar'sRotation,which contain SVNV each year). These will be saved in 95% ethanol and stored in the -20-degree freezer until RNA preparation to determine the geographic distribution of viruses in this species in Alabama.For cotton, populations ofA. gossypiiwill be collected from the field during the flights by Dr. A. Jacobson with help from her team. Populations of no fewer than 100 aphids will be collected from cotton for analysis and stored in 95% ethanolin a -20-degree freezer until RNA preparation.Sequencing:Extraction of total RNA will be conducted usingTrizolandthe protocol available with the purchase of the product.TotalRNAwill be sent for library preparation and sequencing using theNovaSeq6000 for250bp paired-end reads for maximal amounts of sequence present in the insect populations.Virus Analysis:The sequenceswill be analyzed by the construction of a transcriptome.They will also be compared against a database of insect viruses compiled.Virus confirmation:For those viruses present in the insect, it is also possible to identify transcripts of the viromes to determine the possible related relatives.Characterization of bacterial and mycoviruses will also be of interest to further identify their effect on the system.Virus comparisons:Once the viruses have been identified in the aphid and thrips systems, afull comparisonof the similarities and differences between viruses will be conducted. We also plan to compile a database of the virus sequences from insects already sequenced and do direct comparisons to both each other and the newly identified viruses from this study.

Progress 04/01/24 to 03/31/25

Outputs
Target Audience:This project supports the goals of the Pests and Beneficial Species in Agricultural Production Systems program by expanding our understanding of virus-insect associations in key agricultural pests. We have been working to characterize the viromes of Franklinella fusca, Neohydatothrips variabilis, and Aphis gossypii--insects that are common in southeastern agroecosystems and are known vectors of plant viruses. By examining viral communities across both crop and non-crop environments, we uncovered how habitat context and insect biology shape virome composition. These data will provide insight into the potential for virus maintenance and transmission in agricultural landscapes, contributing to a broader understanding of how insect pests interact with viruses beyond known plant pathogens. The findings will inform researchers studying vector ecology and virus emergence, support Extension professionals working on pest and disease management, and provide context for growers aiming to reduce the risk of virus-related yield losses. During this past year, collections were completed for two years of Franklinella fusca on two crop systems for comparison and the sequences were analyzed. We determined that the viral diversity is more significant on a non-crop plant and are currently working on a publication to distribute to the scientific community and have informed extension personnel of our findings. This work concludes that in F. fusca, which in our crop system feeds on peanuts and non-crop clover growing nearby, the community of insects themselves, reflected by the viruses they carry, has a greater diversity in non-crop systems. This implies that the peanuts are conferring selection pressure on the insects themselves and that only a specific population can feed on them in comparison to a non-crop host such as clover. This suggests that the clover is acting as a reservoir for diversity in the viral community. This has greater implications in management. While the reservoir concept seems like a bad idea, it is actually promoting diversity and may reduce selection pressure on insects to change. This reduction could assist with a longer term resistance to the insects in the crop plant. In Neohydatothrips variabilis, we conducted a survey in Auburn in 2023 and sent these for sequencing and discovered that we could identify many members of the viral community previously identified in a study in the midwest in 2018, suggesting a broad geographical range for these viruses. That the greater population of N. variabilis shares many of the same viruses over time and over a broad range. This was communicated to extension personnel to inform growers. However, we also did identify some unique viruses to insects collected in Alabama, which suggests that populations can take on a regional microbiome as well. This could be important and requires further study to understand when and how this occurs. For Aphis gossypii, this is now a collaborative project with a USDA scientist to identify viruses found in this insect and is being headed up by that scientist. A variety of novel aphid viruses were identified in this little-studied insect, and this lab will work towards confirming their presence in our own collections in support of this research. Viromes in insects have generated interest both in the department as it is now clear the viral communities are more diverse than thought and also in the region. Comparative studies have been initiated in surrounding states in response to this work, and a more comprehensive understanding of insect viruses in the southeast will be identified. Changes/Problems:Once sequenced, we noticed that some datasets provide shorter sequences than expected and we are still troubleshooting why that is. We have spoken to colleagues who have made suggestions for improvement, mostly with sample collection and preparation and we are implementing these suggestions in resequencing efforts. We are also investigating if a different sequencing service may be useful, one that provides some of the preparation steps by professionals more used to trouble shooting problems to determine if this results in longer reads from our collections. What opportunities for training and professional development has the project provided?I continue to mentor a graduate student on the project. Through this project she has learned how to interpret and analyze large datasets and with the help of classes at Auburn University has worked through pipelines necessary for understanding viral sequences. This has also enabled training specific to virus sequences and problems which has made the student more specialized, however, still able to use those skills in other capacities. This student also worked with several professors in the department to accomplish her goals, instructing them on the specific issues and problems associated with sequencing viruses in the ecoysystem. I have also been working with a computer science/genetics Undergraduate student to train him to work in bioinformatics more closely and what types of codes are useful for scientists in the field. How have the results been disseminated to communities of interest?We have presented our data at local and regional conferences with a plan to report data to a national conference this summer now that the project is more complete and the data is more understandable. She will present her data to the department as part of her MS defense and we will publish this data this summer. We have submitted sequences of viruses found to Genbank so that others may use our data as well. What do you plan to do during the next reporting period to accomplish the goals?In this next year, we have some repeat sequencing we want to do to determine in some cases why our virus sequences were so short compared to other studies. We also plan on submitted our publications, depositing all relevant sequences into public databases and presenting our data at a national conference.

Impacts
What was accomplished under these goals? Three collections of insects were made for each of the insects listed in Objective one. F. fusca has six populations, four in a diversity on crops study and two as part of a diversity over time study. The diversity on crops study has been completed and the diversity over time is undergoing sequencing now to complete the sequences for comparison. The diversity on crops study is currently being prepared for publication and expected timeline would be to submit during the summer of 2025. N. variabilis was collected for 2023 and sent for sequencing. The analysis of the sequences is completed and the student is currently preparing it for publication. The expected timeline for submission is the summer of 2025. The A. gossypii study evolved into a collaborative project with another lab at Auburn University with a new USDA professor. A survey was conducted and sequenced. Validation of sequences is currently underway and expected publication is in the fall of 2025. This lab also has conducted their own separate collections and sequencing and intends to compare to the existing dataset to determine if any novel viruses have been identified in our collections that warrant their own separate publication. For Objective two- each of the sequences were compared to a larger database on Genbank and determination of similarities and differences were seen. A database of our own sequences was also constructed privately to compare our insects to each other to determine if anything is shared across species in a more direct manner with sequence comparisons. A code was also written to assist with filtering Genbank data to ensure that annotations for viruses are not missed in larger datasets with many of the same sequences which before that had to be filtered by hand. This code identifies and sorts the data and does a secondary assembly specific for these sequences to ensure we have the longest contigs possible for viruses. It was identified that sequence assembly software often does not fully assemble viruses due to the "Quasi-species" problem and this code will address that in part to improve construction of viral contigs in the future for a better comparison of data across the datasets available to us.

Publications


    Progress 04/01/23 to 03/31/24

    Outputs
    Target Audience:The target audience is those interested in the viruses present in insects that may present a problem to either people or agriculture, including but not limited to agriculture scientists, vector entomologists, farmers, etc. Changes/Problems:Initial sample for Aphis gossypii demonstrated lower quality RNA, a new collection will be made to determine if this can be fixed with fresh samples and not stored archival samples. What opportunities for training and professional development has the project provided?Graduate student on this project has learned bioinformatics to analyze insect data. She has also honed her molecular biology skills and continues to learn new ones. She was provided an opportunity to attend the National Entomological Society of America meeting in November to network and present her project. How have the results been disseminated to communities of interest?Presentation at meetings, publication on the work in progress. What do you plan to do during the next reporting period to accomplish the goals?Publish papers and also to attend and present results at meetings.

    Impacts
    What was accomplished under these goals? Insects were collected for all species, including N. variabilis, A. gossypii, and F. fusca. F. fusca has been sequenced and the data initially analyzed, full analysis still pending. Beginning of assembly of the database of all the viruses found in insects so far to compare to those found in our study.

    Publications