Progress 09/01/11 to 08/31/13
Outputs
Target Audience: The target audience for the project findings are the Hawaii agricultural industry, and Hawaii residents and visitors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Although this research project was not intended to provide training and professional development opportunities, the project generated considerable interest with the student employees, and student laboratory assistants. This interest led to five entries over two years into the College Tropical Agriculture and Human Resources and College of Engineering Student Research Symposium (from 2012 and 2013). Entries listed below: 1. Cabalteja C., Thapa P., Kiyabu S., Chun J., Sandall D., Livett B. andBingham J-P.(2013) Expanding the α-Conotoxin Repertoire through Disulfide Bond Permutations. Abstract #6. 25th CTAHR and COE Research Symposium, University of Hawaii, Honolulu,April 12-13. (MS. Student; Poster). 2. Espiritu MJ.andBingham J-P.(2013) Determination of the biochemical effects of naturally produced post translationally modified conotoxins in comparison to synthetic variants. Abstract #13. 25th CTAHR and COE Research Symposium, University of Hawaii, Honolulu,April 12-13. (MS. Student; Poster). 3. Yu P.andBingham J-P. (2013) Cone Snails, Cyclized Peptides, and Fluorophores – A Gateway to Traceable Peptides. Abstract #96. 25th CTAHR and COE Research Symposium, University of Hawaii, Honolulu,April 12-13. (UG. Student; Poster). 4. Cabalteja C.,Kiyabu S.#,Chun J., Sandall D., Livett B. andBingham J-P.(2012) “Challenging the Dogma that Bioactive α-Conotoxins are Globular”. Abstract #28. 24rdCTAHR and COE Research Symposium, University of Hawaii, Honolulu,April 13-14. (MS. Student; Poster). 5. Yu P.#,Thapa P., andBingham J-P.(2012) “Using an Optimized Methodology of TFMSA Cleavage in Peptide Synthesis Bioengineering”. Abstract #93 24rdCTAHR and COE Research Symposium, University of Hawaii, Honolulu,April 13-14. (UG. Student; Poster). How have the results been disseminated to communities of interest? In addition to the 5 professional journal articles, results were presented at the 23rd American Peptide Symposium & 6th International Peptide Symposium, June 22-27, 2013, with a keynote address by Dr. Bingham, and a poster presented by Bergeron et al. Also, a poster, “Bioengineering Peptide Toxins for the Development of Novel Molluscicides and the Protection of Food Crops from Tropical Pathogens in Hawaii,” was presented at the Achievement Rewards for College Scientists (ARCS) Foundation, Scientific Symposium - Poster Session, Honolulu, HI, April 20, 2013. Results were presented to the public through 4 community workshops in November and December 2012. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1 was to identify a method for safely and efficiently removing a human disease-causing pest and its potential hosts from Hawaii produce (e.g. lettuce, herbs, watercress) prior to purchase and/or consumption by Hawaii residents and visitors. The primary objective was to test several possible commercially available sanitizing solutions in order to develop a safe, effective and efficient means of removing the rat lungworm and its intermediate hosts (terrestrial molluscs) from agricultural products prior to their being sold. Thirteen wash solutions were chosen based on their known commercial use on consumer produce and for their potential for removing snails and slugs. Non-native snail species known to be intermediate hosts for rat lungworm and that are common in the Hawaiian Islands were used in the experiments (two size/age classes; juvenile and adult): Succinea tenella, Veronicella cubensis, Deroceras laeve and Parmarion martensi. Each trial consisted of the experimental solutions, fresh produce (whole head of romaine lettuce) and snails. Solutions were prepared per the manufacturer's instructions. Tap water was used as a control. Trials were replicated three times for each age class of each species. For each trial, three snails of the same species and age class were placed in the lettuce; one snail in the center of the lettuce head, another in the inner folds and the third on the outside of the lettuce head. The snails that remained on the produce after the wash and during rinsing were recorded. The results allowed us to evaluate the relative efficacy of the various wash solutions in removing snails and slugs from the produce. The trials indicated that washing alone is not sufficient in removing all snails from produce. Rinsing the whole head of lettuce under cold running water was effective in removing all snails and slugs except those placed in the center of the lettuce, particularly the juveniles. Rinsing each individual leaf under cold running water removed all snails from the produce. These results demonstrate that none of the tested products were any more effective than plain water in removing snails/slugs from the produce. Simple washing in water works as well as any of the tested products, but rinsing individual leaves is necessary to remove all snails/slugs. Since no sanitizing solution was found to be more effective in removing snails/slugs, the project moved forward with phytotoxicity trails of DryTec Calcium Hypochlorite Granular. Phytotoxicity trails included tests of twelve Hawaii-grown commodities, at 3 concentrations (0ppm, 100ppm, and 200ppm), documenting color and wilting for the 7 days post-treatment. For the commodities and treatments tested, there were no phytotoxic effects observed that appeared to be correlated with the DryTec Calcium Hypochlorite Granular treatment. Any observed reduction in quality appeared to be caused by dehydration (wilting), related to an extended time in refrigeration, or possibly improper packaging (and packing) for refrigeration. Although we proposed to start and complete the process of obtaining Hawaii Department of Agriculture clearance for the most effective sanitizing chemicals,we found no solution that was more effective than the others. Project staff shared study results with Hawaii Department of Agriculture to support a review and expansion of the pesticide label for DryTec Calcium Hypochlorite Granular. With the phytotoxicty results, HDOA has prilimary data that supports the expansion of the allowable crops to include varieties grown more frequently in Hawaii. At the conclusion of this project, HDOA had not yet made any changes to pesticide label for DryTec Calcium Hypochlorite Granular. GOAL 2's objectiveswere toexamine the lethality and noxious response of natural and synthetic peptides on invasive snails and slugs that have been determined to carry RLW, and to bioengineer a number of synthetic molluscicide peptides, based on those identified as having potential. Four peptides that demonstrate promise in producing a biodegradable mollusk-specific pesticide have been developed. These peptides are synthetically bioengineered from predatory mollusk that consume other snails. Their isoform pharmacological selectivity in various phyla has been tested, demonstrating specific differentiation between acetylcholine receptor selectivity in worms, fish and mollusk. A number of these peptide candidates with C- to N- terminal peptide backbone cyclization to increase bioavaliability and in vivo stability within the target organism have been bioengineered. Work is on-going to improve synthetic yields and continue with pharmacological refinement to achieved potent activity (100nM Kd) by surface contact or oral absorption. A LAMP assay using a proprietary detection device for Angiostrongylus cantonensis, using cDNA from Chinese worms has been begun to be developed. A novel highly potent peptide toxin was discovered from a carnivorous marine gastropods for pesticide/ molluscicide applications. This peptide has been bioengineered and pharmacologically evaluated for phyla-selectivity (in humans, fish, worms and snails). Present studies indicate potency (~nM range), phyla-genetic ion channel selective (post-synaptic acetylcholine ion channel isoform within gastropods), and to be fully biodegradable - loss of activity due to peptide nature over time. We are now testing its bioavailability in soil, examining breakdown products, as well a continuing its pharmacology assessment to move this peptide compound into field trials in snail control/ eradication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kapono, C.A., Thapa, P., Cabalteja, C.C., Guendisch, D., Collier, A.C., and Bingham, J-P. (2013) Conopeptide truncation as a post-translational modification to increase the pharmacological diversity within the milked venom of Conus magus. Toxicon 70, 170178.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Bergeron, Z.L., Sandall, D.W., Livett, B.G., and Bingham, J-P. (2013) Analysis of Milked Venom from the Mollusc-hunting Cone Snail, Conus textile. Abstract #: 86032; 23rd American Peptide Symposium & 6th International Peptide Symposium, June 22-27, 2013
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Bingham, J-P. Bioactive Peptides from Cone snails: cannibalistic slugs that kill, 23rd American Peptide Symposium & 6th International Peptide Symposium, Hilton Waikoloa Village Big Island, Hawaii, June 22-27, 2013.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Baoanan, Z.G., Milisen, J.W., Slater, D., and Bingham, J-P. (2013) Cone snail aquaculture - A prospect for a biosustainable research commodity. Toxicon.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Cabalteja, C.C., Thapa, P., Sandall, D.W., Kiyabu, S., Chun, J.B., ODonnell, P.A., Livett, B.G., and Bingham, J-P. (2013) Expression of a-conotoxins from Conus virgo and the characterization of a-conotoxin Vr1A. Toxicon.
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
Yeung, N.W., Hayes, K.A., Cowie, R.H., and Hollyer, J.R. In preparation. Unsuccessful removal of snails from produce: Testing 13 vegetable wash solutions. To be submitted to Hawaiian Journal of Medicine and Public Health.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Bergeron, Z.L., Chun, J.B., Baker, M.R., Sandall, D.W., Peigneur, S., Yu, P.Y.C., Thapa, P., Milisen, J.W., Tytgat, J., Livett, B.G., and Bingham,J-P. (2013) A conovenomic analysis of the milked venom from the mollusk-hunting cone shell Conus textile The pharmacological importance of post-translational modi?cations. Peptides 49, 145158.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: In the first project, the goal was to identify a method for safely and efficiently removing a human disease-causing pest and it's potential hosts from Hawaii produce (e.g. lettuce, kale, watercress) prior to purchase and/or consumption by Hawaii residents and visitors. The primary objective of the project was to test several possible commercially available sanitizing solutions in order to develop a safe, effective and efficient means of removing the rat lungworm and its intermediate hosts (terrestrial molluscs) from agricultural products prior to their being sold. Thirteen wash solutions were chosen based on their known commercial use on consumer produce and for their potential for removing snails and slugs. Non-native snail species known to be intermediate hosts for rat lungworm and that are common in the Hawaiian Islands were used in the experiments (two size/age classes; juvenile and adult): Succinea tenella, Veronicella cubensis, Deroceras laeve and Parmarion martensi. Each trial consisted of the experimental solutions, fresh produce (whole head of romaine lettuce) and snails. Solutions were prepared per the manufacturer's instructions. Tap water was used as a control. Trials were replicated three times for each age class of each species. For each trial, three snails of the same species and age class were placed in the lettuce; one snail in the center of the lettuce head, another in the inner folds and the third on the outside of the lettuce head. The snails that remained on the produce after the wash and during rinsing were recorded. The results allowed us to evaluate the relative efficacy of the various wash solutions in removing snails and slugs from the produce. In another project within the same grant, our recently published efforts highlight the potential health issues in dealing with snails. Our direct focus is then to combat both the damage affect of these pests to food crops, as well as control population increases in a focus to provide greater food safety to the consumer by minimizing parasite vector transmission. Four peptides that demonstrate promise in producing a biodegradable mollusk-specific pesticide have been developed. These peptides are synthetically bioengineered from predatory mollusk that consume other snails. Their isoform pharmacological selectivity in various phyla has been tested, demonstrating specific differentiation between acetylcholine receptor selectivity in worms, fish and mollusk. A number of these peptide candidates with C- to N- terminal peptide backbone cyclization to increase bioavaliability and in vivo stability within the target organism have been bioengineered. Work is on-going to improve synthetic yields and continue with pharmacological refinement to achieved potent activity (<100nM Kd) by surface contact or oral absorption. A LAMP assay using a proprietary detection device for Angiostrongylus cantonensis, using cDNA from Chinese worms has been begun to be developed. The final project is to test the phytotoxity of one commercial sanitizer product on a variety of Hawaii produce. A significant delay in getting the sanitizer released from the manufacturer for research purposes delayed the project. PARTICIPANTS: Project Personnel: Jim Hollyer, Program Manager, Department of Plant and Environmental Protection Sciences, University of Hawaii - Principal Investigator. Cathy Tarutani-Weissman, Extension Educator, Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa - provision of products for the experiments to test wash solutions. Work also on legal issues of water sanitizers with the Hawaii Departments of Health and Agriculture. Robert H. Cowie, Researcher, Pacific Biosciences Research Center, University of Hawaii at Manoa - Co Principal Investigator - background on rat lungworm disease and oversight of the experiments to test the wash solutions. Kenneth A. Hayes, Assistant Researcher, Pacific Biosciences Research Center, University of Hawaii at Manoa - experimental design and oversight of the experiments to test wash solutions. Norine W. Yeung, Assistant Researcher, Pacific Biosciences Research Center, University of Hawaii at Manoa - implementation of the experiments to test wash solutions, training of undergraduates. Ashley Kong, Undergraduate Researcher Assistant Pacific Biosciences Research Center, University of Hawaii at Manoa - generation of wash solution experimental data. Deena Gary, Undergraduate Researcher Assistant Pacific Biosciences Research Center, University of Hawaii at Manoa - laboratory assistance. Jon-Paul Bingham, Research Scientist, Department of Molecular Biosciences & Bioengineering, University of Hawaii at Manoa - worked on peptide bioengineering. Vanessa Troegner, Project Logistical Management, Agricultural Development in the American Project, University of Hawaii at Manoa. TARGET AUDIENCES: The goal of this project was to help reduce the probability of humans contracting rat lungworm disease (angiostrongyliasis) through the accidental ingestion of infected snails/slugs on produce by assessing the effectiveness of various commercially available solutions for removing them from produce The aim was not to kill the snails in situ, as recently dead snails may still harbor infective worms, but to identify methods that effectively remove them from the produce. The results will help inform the agricultural industry (e.g. farmers) as well as Hawaii residents and visitors about the most efficient method of removing snails from produce prior to consumption. The impact will be the reduction in possible rat lungworm infections of both residents and visitors, including both children and adults. The value in terms of risk reduction to the Hawaii produce and tourism industries could be in the millions of dollars, and would include potential lost revenues associated with a loss of confidence in the local food production system and in the rejection of products shipped to and from Hawaii. Furthermore, the knowledge gained from this project may save lives in the immediate future. The message that taking apart produce, visually inspecting, and then thoroughly washing/rinsing it to remove snails and slugs has been part of a series of public outreach forums on the islands of Hawaii, Maui and Molokai, with forums on Oahu and Kauai planned for the near future (funded by a different NIFA grant). PROJECT MODIFICATIONS: One Year No Cost Extension of this project was approved; new end date is 8/31/2013.
Impacts
The trials in the first project indicated that washing alone is not sufficient in removing all snails from produce. Rinsing the whole head of lettuce under cold running water was effective in removing all snails and slugs except those placed in the center of the lettuce, particularly the juveniles. Rinsing each individual leaf under cold running water removed all snails from the produce. These results demonstrate that none of the tested products were any more effective than plain water in removing snails/slugs from the produce. Simple washing in water works as well as any of the tested products, but rinsing individual leaves is necessary to remove all snails/slugs.
Publications
- Bingham, J-P., Likeman, R.K., Hawley, J.S., Yu, P.Y.C., and Halford, Z.A. 2012. Conotoxins. In: Manual of Security Sensitive Microbes and Toxins, Ed. D. Liu; CRC Press ISBN:1466553960 (in press).
- Bingham, J-P., Andrews, E.A., Kiyabu, S.M., and Cabalteja, C.C. 2012. Drugs from Slugs, Part II - Conopeptide Bioengineering. Chemico-Biological Interactions, 200: 92-113.
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