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<p>The sterile insect technique (SIT) is an environmental friendly, non-pesticidal control tactic that relies on the mass release of sterile insects to mate with wild insects, thereby producing inviable offspring and reducing pest populations. Irradiation by gamma rays or x-rays is the most common method of inducing the DNA damage needed for sterilization, but irradiated insects suffer from poor performance due to side effects of irradiation, akin to human cancer patients after irradiation treatment. Current programs attempt to compensate for poor post-irradiation performance by releasing enormous numbers of insects, a strategy that drives up economic costs of SIT-based control. My goal is to design, test, and successfully implement a low-oxygen environmental pretreatment that will lower irradiation damage while boosting sterile insect performance in the cactus moth, Cactoblastis cactorum. Enhancing cactus moth post-irradiation performance could reduce the costs and increase the effectiveness of the current control program. Thus my long term goal is to improve sterile cactus moth performance and with it the control efficacy and economy of the sterile insect technique. My hypothesis is that low-oxygen pretreatments will lead to an increase in cellular antioxidant enzymes in cactus moth adults that will reduce irradiation damage due to oxidative free radicals while maintaining sterility. </p>
<p>My main goal is to determine if exposure to low-oxygen pretreatments prior and during irradiation will improve antioxidant capacity and enhance sterile insect performance in the field. Using a combination of antioxidant enhancing pretreatments, I will evaluate which are most likely to improve the cactus moth SIT program. My specific aims are to: </p>
<p>1) Determine whether moths (pupae or adults) exposed to low-oxygen environments prior to and/or during irradiation have enhanced antioxidant defenses. </p>
<p>2) Examine which antioxidant enzymes are involved in the low-oxygen response (superoxide dismutase, catalase, and/or glutathione peroxidase). </p>
<p>3) Evaluate which of the antioxidant-enhancing treatments in #1, lead to better sterile insect performance in the lab by monitoring emergence, longevity, flight, sterility, and mating success. </p>
<p>4) Test whether these antioxidant-enhancing treatments that lead to the best performance in the laboratory, also lead to greater longevity and dispersal during field releases. SIT has been shown to be an effective control tactic for many pest insects, but poor post-irradiation performance limits the efficacy of sterile insects and drives up the costs of SIT programs. If I can show that antioxidant enhancing pretreatments lead to better performance and more effective control in cactus moths, then I can extend this approach to numerous other pests. A long-term outcome of this project is to improve SIT programs for a wide variety of pest insects, allowing American agriculture to meet the growing public demand in the U.S. and Europe for reduced pesticide use.</p>
<p>NON-TECHNICAL SUMMARY:<br/>The cactus moth Cactoblastis cactorum was introduced into the U.S. in 1989 and has expanded to South Carolina, Alabama, Louisiana, and Mississippi. Pest expansion into the western U.S. and Mexico could have devastating effects because collectively the cactus industry is worth about 76 Million US$/year including fruit, pads and dyes. The threat to biodiversity affects 79 species with many of these species being endemic to the US. The sterile insect technique (SIT) is a key component of the current area-wide control plan. SIT floods an area with sterile insects to mate with wild individuals, thereby producing inviable offspring. Ionizing radiation induces sterility by creating double-stranded DNA breaks that produce severe chromosomal damage. Additional radiation damage in other cellular structures results from the production and accumulation of
oxygen radicals and reaction products. This essentially leads to insects with radiation sickness and decreased performance (poor flight ability and decreased mating success). Poor post-irradiation performance is a critical factor limiting the success of all SIT programs. A solution is suggested by my previous work on fruit flies showing that low-oxygen pretreatments increase cellular antioxidant enzymes and post?irradiation fly performance and longevity, while maintaining sterility. In this grant I will use a biochemical perspective to design a series of low-oxygen pretreatments that will enhance post-irradiation cactus moth performance in lab and field assays, while maintaining sterility. Development of performance-enhancing pretreatments for cactus moths meet the broader challenge area in global food security and meets USDA program area priorities in improving food safety and
keeping American agriculture competitive.<p>
APPROACH:<br/>Approach to Aim 1: Using established research protocols and guidelines; I will test a series of low oxygen exposures and durations targeting different stages during the lifecycle of the cactus moth (pupae, pharate adults, adults). To assess antioxidant capacity, insects will be flash frozen in liquid nitrogen as the sampling protocol dictates (0, 0.5, 2, 4, 8, 24, and 48 hrs post treatment) and held at -70C until measured. Insects will be homogenized and antioxidant capacity measured using a common biochemical assay, the decolorization of ABTS. Approach to Aim 2: I will select the three treatments that produced the greatest increases in total antioxidant capacity in Aim 1 for further biochemical dissection. I will measure and quantify the activity of individual antioxidant enzymes using modified commercially available kits for superoxide dismutase (SOD),
catalase and glutathione peroxidase (GPx). Oxidative damage to membranes will be quantified by measuring lipid peroxidation (Tbars) and protein damage will be measured using a modified protein carbonyls assay. Both of these damage assays will allow me to track damage and to determine if oxidative damage following irradiation is decreased following low-oxygen pretreatments. Approach to Aim 3: I will use a series of common quality control metrics developed by the IAEA, FAO, and USDA; which are currently used in sterile insect release programs around the world. I will compare the performance of my various low-oxygen treatments with individuals irradiated in normal oxygen levels to assay for enhanced performance. I will then use survival analyses to determine which pretreatments are most effective for longevity. Second, I will assess mating success and preference via female dissection.
Third, it is critical that our pretreatments enhance post-irradiation performance, but without sacrificing sterility. I will evaluate the sterility of the moths subjected to each pretreatment to ensure that any increase in performance is no also coupled with a loss of sterility by collecting egg sticks and monitoring hatching. Approach to Aim 4: I will perform release and recapture experiments in the field involving several releases over a period of one year to ascertain field performance following standard protocols designed and implemented by Jim Carpenter, Stephen Hight, and other members of the federal cactus moth control program. Release and recapture experiments are the most useful tool for measuring sterile cactus moth performance in the wild and is the best way to determine if my physiologically-guided approach will lead to improved control measures. Specifically, I will chose my
best two performance-enhancing low-oxygen pretreatments from lab bioassays and perform releases in the fall season of year 2 and spring. By comparing recapture rates of the different treatments over a period of three days, we can determine which treatment has the best dispersal and longevity in the field.</p><p>
PROGRESS: <br/>2011/08 TO 2014/07 <br/>Target Audience: During this reporting period my target audience was several undergraduate laboratory assistants that were instructed in biochemistry and lab-based organismal performance assays as it relates to the grant's objectives. Changes/Problems: The last aim if the project, the field experiments, has been delayed due to weather conditions and availability of animals late in 2012. These experiments have been further delayed when I accepted a position at NMSU, and needed several months to move and establish my new lab. Biochemical analysis and field experiments will resume promptly after the successful transfer of the grant from UF to NMSU. What opportunities for training and professional development has the project provided? This project, so far, has provided training opportunities in lab-based biochemical and organismal
performance assays to two post-doctoral associates (besides me), one graduate student, and 4 undergraduate students. Additionally, this project provided training opportunities in field-based experiments for one faculty member, a post-doctoral associate (besides me), and two undergraduate students. This project has provided me with several opportunities for professional development. I have presented research pertinent to this project at 4 national-level conferences, several local conferences, and two invited seminars (including a successful job interview). How have the results been disseminated to communities of interest? Two manuscripts from the work enabled by this grant have been accepted for publication recently. An additional manuscript is in preparation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported
. <br/>PROGRESS: <br/>2012/08/15 TO 2013/08/14 <br/>Target Audience: Nothing Reported. Changes/Problems: I had to request a no cost extension because I needed more time to perform the field release-recapture experiments during late summer and fall 2013 since the field experiments in summer and fall 2012 did not produce the expected outcomes. During the time between the field seasons, I accepted a tenure-track assistant professor position in the department of Biology at New Mexico State University. Currently I am working to transfer the grant from the University of Florida to NMSU, in order to finish the field experiments and analysis. The transfer of the grant will allow me the funds to publish the results and attend a national scientific meeting to disseminate the knowledge. What opportunities for training and professional development has the project provided? Training activities:
I trained two undergraduate students on multiple lab techniques. This training ranged from biochemical analysis to organismal performance assays. My students became proficient sterility and longevity experiments. They also learned data collection, statistical analysis, and presentation skills. 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? I plan to carry out the field release-recapture experiments, therefore completing all the project goals. I also plan to publish two manuscripts on current and ongoing research in the effects of low-oxygen during irradiation in the cactus moth. Additionally I plan to attend a national level scientific meeting to disseminate the results from the field experiment.<br/>
PROGRESS: <br/>2011/08/15 TO 2014/07/14 <br/>Target Audience: During this reporting period my target audience was several undergraduate laboratory assistants that were instructed in biochemistry and lab-based organismal performance assays as it relates to the grant's objectives. Changes/Problems: The last aim if the project, the field experiments, has been delayed due to weather conditions and availability of animals late in 2012. These experiments have been further delayed when I accepted a position at NMSU, and needed several months to move and establish my new lab. Biochemical analysis and field experiments will resume promptly after the successful transfer of the grant from UF to NMSU. What opportunities for training and professional development has the project provided? This project, so far, has provided training opportunities in lab-based biochemical and organismal
performance assays to two post-doctoral associates (besides me), one graduate student, and 4 undergraduate students. Additionally, this project provided training opportunities in field-based experiments for one faculty member, a post-doctoral associate (besides me), and two undergraduate students. This project has provided me with several opportunities for professional development. I have presented research pertinent to this project at 4 national-level conferences, several local conferences, and two invited seminars (including a successful job interview). How have the results been disseminated to communities of interest? Two manuscripts from the work enabled by this grant have been accepted for publication recently. An additional manuscript is in preparation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported.
<br/>PROGRESS: <br/>2011/08/15 TO 2012/08/14<br/>OUTPUTS: In the first year of the funding period, I have been able to meet the first three goals presented in the project narrative. I have determined an appropriate low-oxygen atmospheric treatment and used it in a series of lab-based experiments designed to measure male sexual performance in cactus moths. The accompanied set of biochemistry experiments has also been completed and I am currently performing data analysis in order to write my results for publication. I have had four different undergraduate students involved in the project over the last year. These students took part in different experiments ranging from performance trials to biochemistry. I plan to attend two meetings later this year in which I will present my findings to a broad scientific community. PARTICIPANTS: In the last year, I have had four undergraduate
students involved in the cactus moth project. Their involvement ranged from helping with colony maintenance and treatment preparation to performing biochemical assays on irradiated samples. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.</p>