Source: NEW MEXICO STATE UNIVERSITY submitted to
IMPROVING CACTUS MOTH CONTROL BY ENHANCING STERILE MALE PERFORMANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1002775
Grant No.
2011-67012-21883
Project No.
NM.W-2014-01722
Proposal No.
2014-01722
Multistate No.
(N/A)
Program Code
A7201
Project Start Date
Nov 1, 2013
Project End Date
Aug 31, 2014
Grant Year
2014
Project Director
Lopez-Martinez, G.
Recipient Organization
NEW MEXICO STATE UNIVERSITY
1620 STANDLEY DR ACADEMIC RESH A RM 110
LAS CRUCES,NM 88003-1239
Performing Department
Biology
Non Technical Summary
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 Opuntia industry is worth about 76 Million US$/year including fruit, pads and dyes. The threat to biodiversity affects 79 species. 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 production and accumulation of oxygen radicals and reaction products, essentially leading to insects with radiation sickness and decreased performance. Poor post-irradiation performance is a critical factor limiting the success of all SIT programs. A solution is suggested by my previous work on the fruit flies showing that low-oxygen pretreatments increase cellular antioxidant enzymes and post?irradiation fly performance, 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. I have three mentors, Daniel Hahn at University of Florida, and James Carpenter and Stephen Hight with USDA-ARS.
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
2113110102050%
2113110100050%
Goals / Objectives
Goals for this proposal: My main goal is to determine if exposure to low-oxygen pretreatments prior to irradiation will improve antioxidant capacity and enhance sterile insect performance in the field. Using a combination of antioxidant enhancing pretreatments, I will evaluate which treatments give the most benefit and are likely to improve the cactus moth SIT program. By fully integrating basic lab science with field applications I can truly test whether results achieved in the lab still work in the real world. My specific aims are:1) To determine whether moths exposed to low-oxygen environments prior to and during irradiation have enhanced antioxidant defenses. I will define low-oxygen pretreatments that fit into current rearing protocols that substantially enhance antioxidant capacity. The outcome of this aim will be to use these rapid biochemical assays to prioritize which treatments have the greatest increase in antioxidant capacity and thus should be tested further in laboratory bioassays of organismal performance (Aim 3). My preliminary data shows that 1h of low-oxygen substantially enhances antioxidant capacity (23%) while not affecting survival, and I expect that further improvements will be made with careful parameterization of treatments.2) To determine if low-oxygen pretreatments that lead to enhanced total antioxidant capacity are associated with increases in the activity of three major antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase). I will also determine whether animals exposed to pretreatments that boost antioxidant capacity suffer less post-irradiation cellular damage by assaying for the production of protein carbonyls and lipid peroxides. The outcome of this aim will be to parameterize the physiological responses associated with low-oxygen performance enhancement.3) To evaluate which of the antioxidant-enhancing treatments in Aim1, lead to better sterile insect performance in lab bioassays, including: emergence, longevity, flight ability, sterility, and mating success. I have already identified two treatments that substantially enhance antioxidant capacity (Fig. 2), and therefore Aim 3 is ready to progress even if I do not identify additional treatments with greater antioxidant improvement capacity in Aim 1. The output of Aim 3 will be to test whether treatments that improve antioxidant capacity also produce animals with the best performance in lab bioassays, determining whether antioxidant capacity can be used as a good predictor for whole organism performance.4) To determine if antioxidant-enhancing treatments that produce the best performance in biochemical and standard lab bio-assays, translate to greater longevity and dispersal in field releases. The output of Aim 4 will be to determine if my physiologically-guided approach of boosting antioxidant capacity will lead to real-world improvements in performance that can be directly applied to improving cactus moth control by SIT, and perhaps other SIT programs.
Project Methods
Methods Aim 1: I will test a series of exposures to either anoxia or hypoxia including 1, 1.5, and 2 hours. I will apply these treatments at 4 different time points in the cactus moth lifecycle, illustrated by red arrows in figure 3 above, including 2d and 1d before emergence, and 1d and 2d after emergence. The duration of increased antioxidant capacity will be followed periodically for two days after treatment to estimate how long the effects of low-oxygen treatment will last. Based on the initial results, I will also try 2-4 sequential low-oxygen exposures with the first exposure occurring prior to adult emergence and the second exposure occurring after emergence. I expect these sequential treatments to yield even greater increases in antioxidant capacity than single exposures.For the anoxia pretreatment, pupae or adult moths will be placed in polyethylene bags and flushed with nitrogen for 2 minutes then heat-sealed. For the hypoxia pretreatment the pupae or moths will be heat-sealed in bags allowing the insects to continue consuming oxygen and thereby making the bag hypoxic. I will estimate hypoxia levels in each treatment by using a gas-tight syringe to sample 5 ml of gas from each bagged sample at the end of the treatment period. Oxygen and carbon dioxide levels in treatment bags will be estimated using the manual-bolus integration method (Lighton 2008) employing a respirometry system based on a Li-Cor 7000 infrared CO2 analyzer and a Sable Systems fuel-cell based oxygen analyzer (Oxzilla II).To assess antioxidant capacity, insects will be flash frozen in liquid nitrogen as the time series dictates (0, 0.5, 2, 4, 8, 24, and 48 hrs post treatment) and held at -70°C until the samples are homogenized. Insects will be homogenized in a beadbeater and the supernatant stored at -70°C for analysis. I will use a common biochemical assay, the decolorization of ABTS, to quantify antioxidant capacity (Re et al. 1999).Methods 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 quantify the activity of individual antioxidant enzymes using modified commercially available kits from Cayman Chemical (SOD Assay kit, Catalase Assay kit, Glutathione Peroxidase Assay kit). These are also colorimetric biochemical assays that use co-enzymes or ROS to quantify the amount of each enzyme. For SOD I can further divide the activity between the two enzyme forms (mitochondrial/cytoplasmic), to understand which form responses to the low-oxygen stress.-Oxidative damage to membranes will be quantified using a lipid peroxidation assay (TBARS assay kit). This assay provides a way to record damage and to track its prevention.-Protein carbonyls are the by-product of oxidative stress on proteins. This type of damage can also be quantified with a widely used assay (Levine et al. 1994).Methods Aim 3: Currently there are no quality control/performance metrics for sterile lepidopteran performance, so I will adapt those currently in use for flies to measure the benefits of the pretreatments in cactus moth. I will record emergence, sex ratio, and longevity according to IAEA standards (FAO/IAEA/USDA 2003). In addition I will conduct wind tunnel flight ability tests to insure that the enhanced insects have the best potential for dispersal in the field (Aim 4). Mating success trials will also be conducted to measure mating competence and test for F1 sterility.Methods 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 Carpenter, Hight, and other members of the cactus moth control program (Hight et al. 2005). Release and recapture experiments are the most useful tool for measuring sterile cactus moth performance in the wild (Hight et al. 2005) 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 3 releases in the fall season of year 2 and another 3 releases in the spring. I will use Pherocon 1-C wing traps baited with fertile females (Bloem et al. 2003) to assess male dispersal across a circular grid from a central release point. Each trap will be mounted to a hollow metal stake and placed in a cactus patch at a height of 0.75m. Each of three traps will be separated from each other by 15m in six directions surrounding the site of release (18 traps total). Moths will be marked with different color fluorescent powder (Day-Glo) and released in mid-afternoon (3 to 4pm). By comparing the recapture rates of the different treatments over a period of three days, we can conclude which treatment has the best dispersal and longevity in the field.

Progress 11/01/13 to 08/31/14

Outputs
Target Audience: The target audience for this work consists of fellow scientists involved in basic and applied research, and hopefully government officials in charge of invasive pest control strategies. Additionally, the general consumer (of all economic and social backgrounds) seeking a pesticide-free product make up a big proportion of my target audience. This works provides economically-relevant insight into the effect of low-oxygen hormesis as a low-cost improvement to current environmentally-friendly pesticide-free pest control strategies, like the sterile insect technique. Not only is the efficacy of SIT improved by this method, but the number of insects required for control is less and thus the overall the implementation of this technique should lead to a decrease in pest control costs for commodities that are attacked by invasive species. Essentially the target audience is anyone interested in the improvement of environmentally-friendly non-pesticidal control strategies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project allowed me to manage my own grant, train my own students, and carry out experiments that otherwise I would have not been able to carry out working under someone else’s project. I was given the generous opportunity to move the funding from my post-doctoral position to my new assistant professor position. This allowed me to finish the ongoing research, while training two undergraduate students. I have become proficient in the design of field release studies, a skill I did not previously have. I have also become proficient in circular statistics required to analysis the type of data I collect during the field trials. The research I carried out as part of this project allowed to attain a highly competitive NIH fellowship for a training course in experiment aging at the Aging Institute of the University of Washington. This was both a great training opportunity, but also professional development. I attended at least 9 conferences during the duration of the project and in the last year gave three invited seminar on the work resulting from this project. How have the results been disseminated to communities of interest? The results of this project yielded five publications. Two of which came out in early 2014, two are in review, and the last one will be submitted later this year. The results were also presented at least nice conferences, several of which were international. Four invited seminars have resulted from this work as well. The two published papers gathered a fair amount of news coverage at the local (Gainesville, Florida TV station), state (several Florida TV/radio/news outlets), and national (NPR) levels. The research was feature in at least 10 websites including Science Daily and the Entomological Society of America’s News. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) All objectives proposed in Aim 1 were completed as proposed. Methods Aim 1: I tested exposures to either anoxia or hypoxia including 1, 1.5, and 2 hours. I applied these treatments at 3 different time points in the cactus moth lifecycle (1d before emergence, and 1d and 2d after emergence). The duration of increased antioxidant capacity was followed periodically for two days after treatment to estimate how long the effects of low-oxygen treatment lasted. I had proposed to try sequential low-oxygen exposures, but that was not necessary as single exposure produced the desired outcome. For the anoxia pretreatment, pupae or adult moths were placed in polyethylene bags and flushed with nitrogen for 2 minutes then heat-sealed. For the hypoxia pretreatment the pupae or moths will be heat-sealed in bags allowing the insects to continue consuming oxygen and thereby making the bag hypoxic. I estimated hypoxia levels in each treatment by using a gas-tight syringe to sample 5 ml of gas from each bagged sample at the end of the treatment period. Moths were exposed to hypoxia (low-oxygen) or anoxia (no oxygen) for different periods of time and mortality was assessed. It was determined that one hour of anoxia was the optimal treatment for the rest of the objectives in the project due to the consistent and strong elevation of antioxidant defense. Antioxidant defenses were determined biochemically using the total antioxidant capacity assay, as well as assay for superoxide dismutase, catalase, and glutathione peroxidase. The data was analyzed and we concluded that anoxia led to a 20 to 25% in total antioxidant capacity. This elevation in total antioxidant capacity was an expected change in knowledge which led to a publication and a change in action in Aim 4. 2) All objectives proposed in Aim 2 were completed as proposed. Methods Aim 2: I selected the anoxia treatment, which produced the greatest increase in total antioxidant capacity, for further biochemical dissection. I quantified the activity of individual antioxidant enzymes using modified commercially available kits from Cayman Chemical (SOD Assay kit and Glutathione Peroxidase Assay kit). I adapted an assay for catalase from the literature and used it to quantify levels of this antioxidant enzyme. Oxidative damage to membranes was quantified using the lipid peroxidation assay (TBARS). Protein carbonylation, a measure of oxidative damage to proteins, was quantified using an assay I had previously adapted from the literature (Levine et al. 1994). I carried multiple biochemical assays to determine that in fact superoxide dismutase (SOD) and glutathione peroxidase (GPx) are involved in the elevation of total antioxidant capacity recorded in Aim 1. Catalase was not elevated in response to anoxia. My data clearly shows a pattern in the reduction of oxidative damage to protein and lipids as early as two days after treatment. There is an additional reduction in oxidative damage evident four days after treatment. This aim allowed us to verify some of the mayor gene players behind anoxia hormesis and delivered a change in knowledge on how quickly and prolonged the hormetic effect can be in preventing oxidative damage to proteins and lipids. 3) All objectives proposed in Aim 3 were completed as proposed. Methods Aim 3: I designed and carried out several quality control/performance metrics for sterile lepidopteran performance. Based on input from my mentors who have vast knowledge in this area (James Carpenter and Stephen Hight), I adapted some assays currently in use for flies in order to investigate the effects of anoxia hormesis on cactus moth. I recorded flight ability, mating success, and longevity. Using the information from Aims 1 and 2, a quick a chance in action occurred when I had to redesign flight ability assays. Given that moths that received anoxia were so much active than those irradiated without hormesis, I redesigned the commonly used flight ability test and used one that not only provided flight ability, but also distance and time. This new assay (Lopez-Martinez et al 2014) was reliably repeated and the results were consistent. I found that anoxia prior to irradiation led to an increase in flight propensity, distance flown, and time spent flying. The results also show that anoxia moths are better at mating, and those males live longer. This aim also led to a change in action in the design of the field trial in Aim 4. 4) All objectives proposed in Aim 1 were completed. Methods Aim 4: I performed several release and recapture experiments in the field involving several releases over a period of several weeks following standard protocols designed and implemented by Carpenter, Hight, and other members of the cactus moth control program (Hight et al. 2005). I chose anoxia as the treatment to use in the field studies, given that it has the more robust hormetic response, reduction in post-irradiation oxidative damage, and great improvement in all performance metrics measured. I used Pherocon 1-C wing traps baited with newly developed cactus moth pheromone baits, to assess male dispersal across a circular grid from a central release point. Each trap was mounted to a hollow metal stake and placed in a cactus patch at a height of 0.75m. The original setup was seven transects consisting of nine traps each. Moths were marked with different color fluorescent powder (Day-Glo) and released in mid-afternoon (3:30pm) four times a week for four consecutive weeks. After the unexpected improvement of performance recorded in year 1, we tried again several times in year 2 and 3 with a different trapping design. We extended the trapping area 27-fold and used a circular grid pattern consisting of the levels. Traps were set at 15, 55, and 95 meters from the release point. Sterile male moths were released twice a week for four weeks, but traps were checked every day. Early on in Aim 4, a change in knowledge came when moths release in the field performed just like in the lab. Normally field trials do not mimic lab trials closely. But these sterile male moths flew further and lived longer in the field. This led to a change in action and I redesigned the field trial. Subsequent field trials used a trapping area that was 27 times larger than the original trial. With this new design we were able to collect more anoxia irradiated moths farther away from the release point and days after we no longer collected those moths irradiated without anoxia. This suggests that the effects of hormesis are not an artifact of lab experiments and in fact translate into real world application in a field infested with the pest in question. It is my hope that these results will lead to a chance in condition on how SIT programs incorporate protective treatments and possibly may lead to a more efficient and cost-effective approach to SIT.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: L�pez-Mart�nez G, Carpenter JE, Hight SD, Hahn, DA. Low-oxygen atmospheric treatment improves the performance of irradiation-sterilized male cactus moths used in SIT. Journal of Economic Entomology, 107: 185-197.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: L�pez-Mart�nez G, Hahn DA. Early life hormetic treatments decrease irradiation-induced oxidative damage, increase longevity, and enhance sexual performance during old age in the Caribbean fruit fly. PLOS One, 9: e88128.
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: L�pez-Mart�nez G, Carpenter JE, Hight SD, Hahn DA. Anoxia-conditioning hormesis alters the relationship between irradiation doses for survival and sterility in the cactus moth, Cactoblastis cactorum (Lepidoptera: Pyralidae). Florida Entomologist.
  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: L�pez-Mart�nez G, Meagher RL, Bailey WD, Hahn DA. Low Oxygen atmosphere enhances post-irradiation survival and fertility in the cabbage looper, Trichoplusia ni.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: L�pez-Mart�nez G., Carpenter, J.E., Hight, S.D., and Hahn, D.A. Improving cactus moth SIT: the blending of lab-based hormetic approaches with real world pest control.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: L�pez-Mart�nez G., Visser B, Williams CM, and Hahn DA. Repeated exposures to low-oxygen stress lead to hormetic effects during development and adulthood. Entomological Society of America. Austin, TX.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: L�pez-Mart�nez G., Visser B, Williams CM, and Hahn DA. Repeated anoxia exposures during development have hormetic effects that extend into adulthood. Society for Integrative and Comparative Biology. Austin, TX.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: L�pez-Mart�nez G. Physiological conditioning hormesis improves post-irradiation performance in young and aging flies. The 13th annual international conference on dose-response. Amherst, MA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: L�pez-Mart�nez G. Effect of free radical damage and hormesis on lifespan and immunity. NIH National Institute of Aging (NIA) Summer training course in experimental aging. University of Washington, Seattle, WA.