Source: UNIV OF HAWAII submitted to
POST-HARVEST MANAGEMENT OF SLUGS AND SNAILS POTENTIALLY CARRYING RAT LUNGWORM (ANGIOSTRONGYLUS CANTONENSIS) IN HAWAII.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0227000
Grant No.
2011-37610-31162
Project No.
HAW01955-G
Proposal No.
2011-05297
Multistate No.
(N/A)
Program Code
NI
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2013
Grant Year
2011
Project Director
Hollyer, J.
Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
Plant & Environmental Protection Sciences
Non Technical Summary
Residents of the state of Hawaii are experiencing an increase in the number of eosinophilic meningitis cases. In the last 8 years, there have been 41 cases, either confirmed or probable, and many consider these cases under reported. The cause of these outbreaks is the Rat Lungworm (RLW), Angiostrongylus cantonensis, a parasitic nematode that lives in the lungs of rats and that have slugs and snails as intermediate hosts. Slugs and snails can often be found on commercial farms and home gardens in Hawaii and most growers are not aware of the dangers of not managing these pests. Currently, there are no commercial water sanitizers that have been studied or Environmental Protection Agency-listed as an approved pesticide to kill slugs and snails in produce wash water. Therefore, this project will investigate the efficacy of at least 12 commercial water sanitizers on their ability to kill the Angiostrongylus cantonensis nematode and at least 13 known slug, snail and flat worm hosts of RLW. The phototoxicity of each sanitizer that is found to be effective for killing snails will also be tested on leafy greens so that any postharvest damage can be identified and managed. The best chemicals will then be chosen to complete the approval process from the Hawaii Department of Agriculture for use in commercial production. In addition, this project will advance work on new molluscicides that could potentially target certain species of slugs and snails, and yet prove harmless to other animals should they be consumed.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7126099302040%
7223130106030%
2141499115030%
Goals / Objectives
GOAL 1's objectives are: 1. To test several commercially available sanitizing solutions that could be developed as a safe, effective and efficient means of removing the RLW and its intermediate hosts (snails and slugs) from agricultural products prior to their being sold locally and/or exported from Hawaii. 2. To start and complete the process of obtaining Hawaii Department of Agriculture clearance for the most effective sanitizing chemicals. 3. To do an observational study on the potential phytotoxic effects that the best sanitizers might have on leafy greens. GOAL 2's objectives are: 1. To examine the lethality and noxious response of natural and synthetic peptides on invasive snails and slugs that have been determined to carry RLW. 2. To bioengineer a number of synthetic molluscicide peptides, based on those identified as having potential in objective 1, and to assess the improvement, if any, of the lethality of these peptides compared to the natural and synthetic peptides evaluated in Goal 1.
Project Methods
GOAL 1 - Objective 1, 2 and 3: water sanitizer testing for lethality, postharvest quality and registration. 1. This objective will be met through a series of replicated trials involving commercially available washes, local produce and different size classes of several RLW snail/slug hosts common in agricultural settings. Trials will be done by placing 6 RLW hosts (2 of each size class; <5 mm, 5-10mm, >10 mm) on produce. The RLW hosts will be allowed to acclimate, and then the produce will be washed for a set period of time in the sanitation solution, and at 7 concentrations (1-30 ppm). Each concentration will be tested 5 times per species, for a total of 35 trials per species for each solution. Effectiveness will be determined by number of hosts remaining on the produce and their continued survival after exposure. In addition, the RLW nematode, outside of the host, will be subjected to the sanitizers and the results recorded. 2. Discussions with subject matter experts (SME) help determine the most efficacious chemicals made legally available to Hawaii farmers. SMEs include scientists from the Hawaii's Departments of Agriculture and Health, and other federal and state public health agencies. 3. Produce that slugs and snails typically inhabit will be purchased and subjected to the most effective sanitizers and any phototoxic effects will be observed. This information will inform the outreach material that is produced. GOAL 2 - Objectives 1 and 2: new molluscicide development and testing 1. Peptide lethality application - Natural isolated and synthetic Reverse-High Pressure Liquid Chromatography (RP- HPLC) purified peptides will be dissolved in water and drops (5-10microL in vol.) will be placed on live snails/slugs. Activity will be monitored for 36 hrs, with lethal concentrations (LD50) being calculated in peptide concentration/kg of body weight. 2. Assessment of nociceptor response in live snails/slugs will be undertaken to determine boundary/confinement abilities of native and synthetic isolated venom peptides. Large Whatman filter papers will be divided in thirds with 1/3 segment of the paper removed. Peptide material, varying concentrations (i.e. 10nM - 50microM), will be evenly diffused onto the filter paper by soaking and then allowed to air dry. Impregnated filters will be placed into low-walled glass lidded Petri dishes of the same diameter. Water will be atomized to surface of Petri dish/filter paper prior to introduction of slug/snail. Simple observations on slug/snail movement and behavior will be digitally monitored for 24 hr to determine the presence of avoidance behavior. Establishment of normal movement patterns will be provided by water controls. 3. Synthetic peptide candidates, both native-like and bioengineered peptides, most of which are already in isolated and purified forms, will be examined to improve bioavailability by the thiol-ester ligation of the N- to C- terminal of the peptides using established 'in-house' methods. This approach will require re-synthesis of selected peptide candidates by Solid Phase peptide synthesis, again using established 'in-house' techniques.

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 &alpha;-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 &ndash; 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) &ldquo;Challenging the Dogma that Bioactive &alpha;-Conotoxins are Globular&rdquo;. 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) &ldquo;Using an Optimized Methodology of TFMSA Cleavage in Peptide Synthesis Bioengineering&rdquo;. 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 &amp; 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, &ldquo;Bioengineering Peptide Toxins for the Development of Novel Molluscicides and the Protection of Food Crops from Tropical Pathogens in Hawaii,&rdquo; 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&#39;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&#39;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.


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.