Source: UNIVERSITY OF ARIZONA submitted to
DEVELOPMENT OF STABLE ISOTOPES FOR IDENTIFICATION OF STERILE INSECTS AND PEST ORIGIN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1004004
Grant No.
(N/A)
Project No.
ARZT-1361110-H31-166
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2014
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Davidowitz, G, .
Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Entomology
Non Technical Summary
The USDA uses numerous methods to control agricultural pests. One of these is Sterile Insect Technology (SIT) in which massive numbers of sterile insects are released in a problematic area. These sterile insects mate with the naturally occurring pests, but produce no offspring leading to declines in the pest population or its extinction. For this technology to work it is essential to be able to distinguish between the released sterile insects and those that occur naturally. Mistaking sterile released insects for wild-target pests can trigger costly response measures or delay eradication declarations in programs that use SIT as a primary control measure. This project will develop and test the efficacy of using carbon stable isotope technology to distinguish between SIT reared and wild pests. The SIT reared pests will be reared on a diet with a different nonradioactive stable carbon isotope signature than that of the host plant. Pests will be collected in the field and tested whether their isotope signature is that of the host plant or the SIT reared pests. This project will test this technology on four pest species: the pink boll worm (PBW), light-brown apple moth (LBAM), Mediterranean fruit fly (med fly) and Mexican fruit fly (mex fly). In addition, this project will develop the technology of combining isotope signatures with fatty acid profiles to determine the source of origin and host identity of invasive pests.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21131101130100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
1) Develop new Isotopic methods and tools to allow Identification and determination of sterile and wild pest insects collected on monitoring traps in APHIS·PP sterile insect technique (SIT) release programs for eradication of pink bollworm (PBW), Mediterranean fruit fly and Mexican fruit fly.2) Develop and apply new isotopic and fatty acid analysis tools for identification and to trace back probable origin and host identity of invasive plant pests found in CAPS programs or in eradication or area-wide control program monitoring.
Project Methods
Goal l: Development and testing of Isotopic methods for Identification of sterile insects. In the first year, 50 each of wild and factory reared insects: of Mediterranean fruit fly, PBW, and LBAM will be prepared for isotopic analysis of carbon, nitrogen and non-exchangeable deuterium throughout the field season to determine which Isotopes provide the best means of separating mass-reared sterile insect from wild field collected samples. In the case of medfly this will focus on disseminating and transferring the technology and highlighting the programmatic benefits of using the previously tested carbon Isotope signatures for distinguishing mass reared from wild populations in critical cases (Hood et al., 2009). For the wild insects, the larval host plant species will be recorded (if available) to determine if different host plant types influence isotopic signals.To compare the relative performance of the Picarro carbon analysis machine to stable isotope mass spectrometry, an additional sample of 50 each of wild and mass-reared PBW and LBAM moths and 50 each of Mediterranean fruit flies will be tested at the University of Arizona, Department of Entomology to determine the ratios of 12C to 13C of wild vs. factory reared for each type on insect. For analysis of the effect of glue on samples isotope ratios, LBAM will be used as a model to test the effects of the soft versus hard glue on measurement of carbon isotope ratios and to test methods for cleaning samples of soft glue.Wild moths will be collected from the field in Arizona and California for PBW and from California for mass-reared PBW and LBAM will be provided from laboratory insectaries maintained by USDA·APHIS· PPQ in Arizona and California. Wild and factory medfly will be collected by our cooperators from Guatemala and from the California Dept. of Food and Agriculture medfly emergence facility in Los Alamitos, CA. Factory produced moths and medfly will be reared on factory diet made with C4 cane sugar.If positive results are obtained in the first year, in year two the Picarro instrument will be installed in one of our operational program laboratories such the CDFA Diagnostics laboratory in Sacramento, CA or the pink bollworm facility in Phoenix, AZ for routine testing and sample cross comparisons (Picarro vs. Isotope-ratio mass spectrometry= IRMS). Protocols for sample preparation, coding, shipping and documentation will be written and established. This will include further testing of sticky traps and sample clean up from sticky trap collected samples. A training course in sample preparation, data interpretation and methodologies will be carried out.In year three, continued IRMS based analysis and technical support will be provided leading to development of independent hub of stable isotope analysis and expertise within the laboratories of our cooperators in California, Florida, Texas, Arizona, New Mexico, and APHIS.A key component of this project will be to provide support for capacity building and technology transfer in the use of isotopic techniques to PPQ and its stakeholders so that the technology can be integrated into our cooperative programs for area-wide eradication and control against fruit flies and pink bollworm and against other invasive pests targeted in CAPS programs. The use of these tools, while well established in ecological studies, have to date not been adopted for use by any phytosanitary regulatory surveillance or eradication programs. These new tools will assist in making timely decisions to determine regulatory responses and to provide new methods for determining the source of new pest incursions. This would be achieved by a first year of initial methods and development testing and followed by technological transfer, training and support offered by Rebecca Hood-Nowotny in years two and three.Goal 2: Development of stable isotope and fatty acid analysis for determining origin and host. Our hypothesis is that fatty acid and isotope profiles of insects are dependent on their natal host species and it is possible to reliably trace back the identity of the natal host and gain information on its larval host plant and its origin (local vs. overseas or distant region).

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of lab technician, Heather Costa, in use of picarro isotope analyzer 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?Continue analysis of samples

Impacts
What was accomplished under these goals? Carbon Stable Isotope Analysis using CM-CRDS Picarro System Insect samples for isotope analysis that were collected from the field and/or reared in the laboratory were received and processed for Carbon isotope signature analysis using the Picarro CM-CRDS system. Since the last report, more than 500 samples of insect and plant tissue have been processed using the Picarro System. The results have been entered into a database, and are currently being statistically analyzed. The following table shows the samples processed since the last report. Fatty acid analysis for determining host origin We previously reported that Fatty acid fingerprint analysis of wild Mexican Fruit fly (Mexfly), Anastrepha ludens, subsequently reared from guava, mango, or sugarcane bagasse artificial diet showed clear differences between fly types. This suggests this method could be a useful tool in determining the host origin of the flies caught on the traps. Given that there are a number of laboratories that have GC-FID equipment for fatty acid analysis as opposed to the rarer Py-GCMS systems which we initially envisaged that we conduct the project on, we decided that we should develop a simple, fast and reliable method for fatty acid extraction and analysis for insects as opposed to concentrating on the Py-GCMS method. We will, however, continue to compare data to the Py-GCMS data. We have streamlined the fatty acid methodology to take advantage of a number of developments in laboratory equipment e.g. tabletop centrifuges and the availability of chemical resistant micro-centrifuge tubes etc. Given that we have a wealth of fatty acid analysis expertise available at AIT we thought it best to draw on this expertise to develop a simple to implement, universally applicable method of FA extraction, which can be conducted in less than 24 hours. The new method we have established has changed to more commercially available reagents, has less transfer steps, and the method is simpler to carry out without error. The data shown below demonstrate that it is possible to distinguish the different flies depending on the diets they consumed, and it is clear that we have dietary routing. This can be confirmed irrespective of the method used to analyze the patterns. From these data it can be shown that the two methods of fatty acid extraction yield independently valid, but incomparable results. This is a consequence of the annotation of the different fatty acids by the two different systems, as we used different CG columns and injection temperatures. Although we use standards throughout both methods, one method is driven by library annotation and the other is driven by standard comparison. Because each system uses different columns with different retention times, a fatty acid may appear as a single variant in one system but chromatographically separate into two distinct fatty acids in the other system, added to this is the problem of cis-trans isomers. This makes cross comparison difficult and results in loss of information if one standardizes back to of the other methods. We will attempt to overcome this by sending a sample of our lab standard to the Linz laboratory and standardizing back to that this should allow us to make a rigorous comparison. The new Fatty acid extraction method for the GC-FID proved robust and simple to implement. We can now easily analyze over 20 samples per day, with a one-day analysis time. We need to further determine our detection limits of the new extraction system. However, all insect samples submitted have been prepared and analyzed, we still need to run isotope and FA analysis on the diets but we had put this off until we had the method extraction established as it is not a trivial task and there was minimal sample. See guiding operational procedure. All the Py-GC MS samples have been analyzed and we are slowly going through the procedure of standardization and data analysis.

Publications


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training for a technician, Heather Costa, in the use of the Picarro carbon stable isotope analyzer. 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?Goal 1: Develop and test stable isotopic methods for identification of sterile (mass reared) insects for possible inclusion into PPQ sterile insect release programs. Test Objective 1) Determine isotopic differences for carbon, nitrogen, sulfur and non-exchangeable deuterium between wild and mass-reared insects for Medfly, Mexfly and LBAM as a model insect using both IRMS mass-spec and the Picarro machine for carbon isotopes. Test Objective 2) For carbon isotopes, determine the minimum sample mass needed and optimal body part (e.g., leg, wing, abdomen, and thorax) for reliable and repeatable stable isotope analysis using both the Picarro and IRMS machines for isotope analysis for wild and mass-reared insects for Medfly, Mexfly, and LBAM. Test Objective 3) Determine factors that may influence reliability of stable isotope signal, insect age and dispersal, adult feeding, trap glue type. Test Objective 4) Determine if deuterium and other isotope signals can be used to identify the provenance of Asian gypsy moth eggs collected on container ships arriving at US ports and determine if there are unique isotopic signatures associated with specific ports or countries. Goal 2: Develop stable isotope and fatty acid analysis tools for determining invasive pest origin and natal host. Test Objective 5) Determine the specificity of using individual insect fatty acid analysis for identifying natal host crop species for oriental fruit fly, Lobesia botrana, Medfly, Mexfly, PBW and LBAM. Test Objective 6) Combine results from isotope analysis and fatty acid signature analysis using Bayesian statistical methods for development of simple identification of the most probable natal host species and locations. Test Objective 7) Develop a protocol for use of stable isotope tools and fatty acid analysis methods for determination of wild vs. sterile insects and natal host origin for APHIS program pests: PBW, Medfly, Mexfly, oriental fruit fly, LBAM and Lobesia botrana.

    Impacts
    What was accomplished under these goals? Carbon Stable Isotope Analysis using CM-CRDS Picarro System Insect samples for isotope analysis that were collected from the field and/or reared in the laboratory were received and processed for Carbon isotope signature analysis using the Picarro CM-CRDS system. Since the last report, more than 500 samples of insect and plant tissue have been processed using the Picarro System. The results have been entered into a database, and are currently being statistically analyzed. The following table shows the samples processed since the last report. 2)Fatty acid analysis for determining host origin We previously reported that Fatty acid fingerprint analysis of wild Mexican Fruit fly (Mexfly), Anastrepha ludens, subsequently reared from guava, mango, or sugarcane bagasse artificial diet showed clear differences between fly types. This suggests this method could be a useful tool in determining the host origin of the flies caught on the traps. Given that there are a number of laboratories that have GC-FID equipment for fatty acid analysis as opposed to the rarer Py-GCMS systems which we initially envisaged that we conduct the project on, we decided that we should develop a simple, fast and reliable method for fatty acid extraction and analysis for insects as opposed to concentrating on the Py-GCMS method. We will, however, continue to compare data to the Py-GCMS data. We have streamlined the fatty acid methodology to take advantage of a number of developments in laboratory equipment e.g. tabletop centrifuges and the availability of chemical resistant micro-centrifuge tubes etc. Given that we have a wealth of fatty acid analysis expertise available at AIT we thought it best to draw on this expertise to develop a simple to implement, universally applicable method of FA extraction, which can be conducted in less than 24 hours. The new method we have established has changed to more commercially available reagents, has less transfer steps, and the method is simpler to carry out without error. The data shown below demonstrate that it is possible to distinguish the different flies depending on the diets they consumed, and is it is clear that we have dietary routing. This can be confirmed irrespective of the method used to analyze the patterns. From these data it can be shown that the two methods of fatty acid extraction yield independently valid, but incomparable results. This is a consequence of the annotation of the different fatty acids by the two different systems, as we used different CG columns and injection temperatures. Although we use standards throughout both methods, one method is driven by library annotation and the other is driven by standard comparison. Because each system uses different columns with different retention times, a fatty acid may appear as a single variant in one system but chromatographically separate into two distinct fatty acids in the other system, added to this is the problem of cis-trans isomers. This makes cross comparison difficult and results in loss of information if one standardizes back to of the other methods. We will attempt to overcome this by sending a sample of our lab standard to the Linz laboratory and standardizing back to that this should allow us to make a rigorous comparison. The new Fatty acid extraction method for the GC-FID proved robust and simple to implement. We can now easily analyze over 20 samples per day, with a one-day analysis time. We need to further determine our detection limits of the new extraction system. However all insect samples submitted have been prepared and analyzed, we still need to run isotope and FA analysis on the diets but we had put this off until we had the method extraction established as it is not a trivial task and there was minimal sample . See guiding operational procedure. All the Py-GC MS samples have been analyzed and we are slowly going through the procedure of standardization and data analysis.

    Publications

    • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Hood-Nowotny, R., L. Mayr, J. Heindler, N. Saad, R. Seth, G. Davidowitz, G. Simmons. (2016). Insect isotope analysis using cavity ring-down spectroscopy: Moving towards incorporating isotope analysis into area-wide management program. Florida Entomologist (in press).