Source: UNIV OF MASSACHUSETTS submitted to
DEVELOPMENT OF A BACULOVIRUS FOR WINTER MOTH IPM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
SPECIAL GRANT
Reporting Frequency
Annual
Accession No.
0221979
Grant No.
2010-34103-21214
Project No.
MAS00201002776
Proposal No.
2010-02776
Multistate No.
(N/A)
Program Code
QQ.NE
Project Start Date
Sep 1, 2010
Project End Date
Aug 31, 2013
Grant Year
2010
Project Director
Elkinton, J. S.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Microbiology
Non Technical Summary
The winter moth, Operophtera brumata, is an invasive pest in a large portion of the Northeastern US including Massachusetts and Rhode Island. The expanding range and lack of adequate non-chemical control measures has lead to identification of the development of integrated pest management (IPM) tactics for the control of this pest by the Pest Management Alternatives Program as a priority in the in Northeastern region. Based on successful attempts to control this insect in Nova Scotia using the tachinid parasitoid, Cyzenis albicans, we have initiated releases of this fly in Massachusetts to control winter moth. These releases have had limited success. In order to augment this biological control agent, we proposed to test the ability of the naturally occurring baculovirus O. bumata nuclelopolyhedrovirus (OpbuNPV) to become established in pest populations by introducing this agent into blueberry patches infested with winter moth. The naturally occurring biopesticide OpuNPV which we have recently found MA, is specific for O. bumata and can therefore be used with C. albicans in an IPM program to control winter moth without having a deleterious effect on other insect species including insect pollinators like honey bees. The first step in the development of this virus as a biological agent, for use as part of an integrated control program for winter moth, will be the identification of more virulent stains of the virus.
Animal Health Component
(N/A)
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21631101130100%
Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
The goal of the proposed work is to select an Operophtera bumata nuclelopolyhedrovirus (OpbuNPV) isolate with increased virulence for winter moth. Currently we have geographic isolates of OpbuNPV from MA, the U.K. and Norway, and expect to recover additional isolates from France and Austria and other parts of Europe. Using winter moth larvae from MA, we plan to perform bio-assays with each of these isolates to determine which, if any, are more potent against insects found here in the Northeast. In addition, we plan to select for a sub-population of faster killing viruses from each of these geographic isolates. It is very likely that the MA isolate is well suited for use in controlling insects in the region, however if we are able identify a more potent isolate we plan to incorporate that in field tests to be conducted later. Isolates found to have enhanced virulence compared to MA virus, as determined by laboratory bio-assays will then be tested in future field experiments to examine their effectiveness as potential biological control agents for use in controlling winter moth. In this way we hope to identify virus isolates with different properties and compare the performance of these viruses in field tests.
Project Methods
More virulent, faster killing OpbuNPV isolates from each of our geographic isolates will be obtained utilizing 9 to 12 cycles of selection following a regimen of collecting OBs from insects that die the soonest after infection as a source of the inoculum for a subsequent round of infection. The potency of these virus isolates selected for a faster speed of killing will then be compared to that of our other geographic isolates in bioassays. Any of these isolates showing enhance virulence in our laboratory will then be incorporated into our field trials Both the potency (LD50) and the speed of kill (LT50) for each of the virus isolates will be determined in laboratory bioassays using larvae from our stock of laboratory reared insects. For the lethal dose (LD) assays newly emerged third instars will be starved for 4-6 hrs then place on individual diet cubes to which a specific dose of virus has been added. Five virus doses ranging from ~ 10 to 1000 OBs per diet cube will be used with 20 to 30 larvae per dose. Virus dilutions will be made in water containing blue food coloring which will allow us follow consumption and passage of the inoculum through the insects which will appear blue upon ingestion of dye and excrete blue frass. Insects which do not consume the complete diet cube within 30 minutes will be excluded from the test. Three replicates of this experiment will be performed for each of the viruses to be tested. LD50 values for each isolate will be determined by probit analysis and differences in LD50s will be determined using lethal dose ratio methods. The same methods will be used to measure the mean survival time (LT50) of insects infected with the different isolates using virus dose for each isolate that results in ~ 90% mortality (LD90). Differences in mean survival times will be estimated by the Log-Rank test.

Progress 09/01/10 to 08/31/13

Outputs
Target Audience: Nothing Reported 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? Project terminated due to PI retirement

Publications


    Progress 09/01/11 to 08/31/12

    Outputs
    OUTPUTS: The results from our research have stimulated interest and collaboration with Dr. Robert Harrison of the USDA Invasive Insect Biocontrol & Behavior Lab in Beltsville MD. In collaboration with Dr. Harrison, we have begun studies to examine the susceptibility of winter moth (Operophtera Brumata) to infection by several other nucleopolyhedroviruses. We have also initiated efforts to establish a winter moth cell line for future virus studies. PARTICIPANTS: Individuals: PD. Joseph S. Elkinton,University of Massachusetts- Amherst CoPD John P. Burand, University of Massachusetts - Amherst Training: Postdoctoral Associate: Woojin Kim, Graduate Student: Ajanta De Collaborators: Heather Faubert, Department of Plant Sciences, University of Rhode Island Robert Harrison of the USDA, Beltsville MD TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: It has proven to be difficult to generate enough high quality winter moth larvae to conduct virus bio-assays that produce reliable results. Therefore, we have focused on qualitative measurements of difference in the level of virulence of each viral isolate. We have also decided to focus on in vitro cultivation and genetic characterization of different OpbuNPV isolates and viruses from other related insect species.

    Impacts
    We have improved methods for scaling up rearing of winter moth larvae for virus production and bioassays. This method was used for the production of virus samples from Massachusetts, Norway, British Columbia and the United Kingdom for use in bioassays to compare their activity. Multiple sequence alignments of the polyhedrin and p74 genes of OpbuNPV isolates from Massachusetts, Norway, British Columbia and the United Kingdom were performed using MUSCLE, and then phylogenetic analysis of these sequences was performed using a maximum parsimony method with MEGA5 computer software. Both the polyhedrin and p74 sequences made distinctive clades according to their geographic collection sites. In almost every case each geographic isolate was localized in the same clade. For the polyhedrin gene the MA clade is closer to the clade of the other Norway isolates than any of the other clades. The p74 gene showed very similar trend to that observed for the polyhedrin tree. Each clade represented geologically segregated virus isolates. Interestingly, for p74 a small group of MA isolates made a clade closer to the clade of UK isolates.

    Publications

    • Burand, J.P., W. Kim, A. Welch, and J.S. Elkinton. 2011. Identification of a nucleopolyhedrovirus in winter moth populations from Massachusetts. J. Invertebr Pathol. 108(3):217-9.


    Progress 09/01/10 to 08/31/11

    Outputs
    OUTPUTS: The results from our laboratory research were presented at the 44th annual meeting of the Society for Invertebrate Pathology in Halifax, Nova Scotia, August 2011 in two posters entitled: Possible Origin of a Nucleopolyhedrovirus in Winter Moth Populations in Massachusetts. PARTICIPANTS: Individuals: PD. John P. Burand, University of Massachusetts - Amherst CoPD Joseph S. Elkinton, University of Massachusetts- Amherst Training: Postdoctoral Associate: Woojin Kim, Graduate Student: Ajanta De Collaborator: Heather Faubert, Department of Plant Sciences, University of Rhode Island TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    By using a diagnostic PCR to amplify a region of the baculovirus polyhedrin gene, we were able to detect the Operophtera. brumata nucleopolyhedrovirus (OpbuNPV) in winter moth larvae collected from field sites in Massachusetts. This represents the first documented report of the winter moth virus, OpbuNPV in populations of the insect in the U.S. A total of 67 larvae from 5 collection sites in Massachusetts were analyzed and a total of 19 or ~28% were positive for OpbuNPV DNA. The infected larvae were found in only 2 sites of which on average 37.5% were found to contain viral DNA sequences. Interestingly, 6 of the 59 pupae collected and analyzed were found to be positive for viral DNA sequences. This represents the first documentation of baculovirus infections being found in pupae collected from field populations, and possibly reflects the occult nature of this virus.

    Publications

    • Burand, J.P., W. Kim, A. Welch, and J.S. Elkinton. 2011. Identification of a nucleopolyhedrovirus in winter moth populations from Massachusetts. J. Invertebr Pathol. 108(3):217-9.