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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.<P> 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.
Non-Technical Summary: <BR>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. <P> Approach: <BR> 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.