Neuroangiostrongyliasis, caused by Angiostrongylus cantonensis , has been reported in Hawaiʻi since the 1950's. An increase in cases is being reported primarily from East Hawaiʻi Island, correlated with the introduction of the semi-slug Parmarion martensi . Households in areas lacking infrastructure for water must use rainwater catchment as their primary domestic water supply, for which there is no federal, state, or county regulation. Despite evidence that slugs and snails can contaminate water and cause infection, regulatory bodies have not addressed this potential transmission route. This study evaluates: 1) the emergence of live, infective-stage A . cantonensis larvae from drowned, non-native, pestiforous gastropods; 2) larvae location in an undisturbed water column; 3) longevity of free-living larvae in water; and 4) effectiveness of rainwater catchment filters in blocking infective-stage larvae. Larvae were shed from minced and whole gastropods drowned in either municipal water or rainwater with ~94% of larvae recovered from the bottom of the water column 72–96 hours post drowning. Infective-stage larvae were active for 21 days in municipal water. Histological sectioning of P . martensi showed proximity of nematode larvae to the body wall of the gastropod, consistent with the potential for shedding of larvae in slime. Gastropod tissue squashes showed effectivity as a quick screening method. Live, infective-stage larvae were able to traverse rainwater catchment polypropylene sediment filters of 20 μm, 10 μm, 5 μm, and 1 μm filtration ratings, but not a 5 μm carbon block filter. These results demonstrate that live, infective-stage A . cantonensis larvae emerge from drowned snails and slugs, survive for extended periods of time in water, and may be able to enter a catchment user's household water supply. This study illustrates the need to better investigate and understand the potential role of contaminated water as a transmission route for neuroangiostrongyliasis.

16 Neuroangiostrongyliasis, caused by Angiostrongylus cantonensis, has been reported in Hawaiʻi 17 since the 1950's. An increase in cases is being reported primarily from East Hawaiʻi Island, 18 correlated with the introduction of the semi-slug Parmarion martensi. Households in areas 19 lacking infrastructure for water must use rainwater catchment as their primary domestic water 20 supply, for which there is no federal, state, or county regulation. Despite evidence that 21 contaminated water can cause infection, regulatory bodies have not addressed this potential 22 transmission route. This study evaluates: 1) the emergence of live, infective-stage A. cantonensis 23 larvae from drowned, non-native, pestiforous gastropods; 2) larvae location in an undisturbed 24 water column; 3) longevity of free-living larvae in water; and 4) effectiveness of rainwater 25 catchment filters in blocking infective-stage larvae. Larvae were shed from minced and whole 26 gastropods drowned in either municipal water or rainwater with >94% of larvae recovered from 27 the bottom of the water column. Infective-stage larvae were active for 21 days in municipal 28 water. Histological sectioning of P. martensi showed proximity of nematode larvae to the body 29 wall of the gastropod, consistent with the potential for shedding of larvae in slime. Gastropod 2 30 tissue squashes showed effectivity as a quick screening method. Live, infective-stage larvae were 31 able to traverse rainwater catchment polypropylene sediment filters of 20 µm, 10 µm, 5 µm, and 32 1 µm filtration ratings, but not a 5 µm carbon block filter. These results demonstrate that live, 33 infective-stage A. cantonensis larvae can and do emerge from drowned snails and slugs, survive 34 for extended periods of time in water, and that the potential exists that they enter the household 35 water supply. This study illustrates the need to better investigate and understand the potential 36 role of contaminated water as a transmission route for neuroangiostrongyliasis. 37 38 Introduction 39 The nematode Angiostrongylus cantonensis is established throughout the main Hawaiian Islands 40 with the possible exception of Lānaʻi [1, 2, 3]. The complex lifecycle of this parasite has been 41 well-described in the literature [4, 5, 6, 7]. In Hawaiʻi, Rattus rattus and Rattus exulans are 42 important definitive hosts, and many gastropod species are effective intermediate hosts including 43 Achatina fulica, Euglandia rosea, Laevicaulis alte, Limax maximus, Parmarion martensi and 44 Veronicella cubensis [1, 2, 8]. The third stage larva (L3) is harbored in the intermediate host, and45 it is this larval stage that is infective to rats and accidental hosts, including humans, as the L3 46 larvae can safely pass through the acidic environment of the mammalian gut. There are also 47 paratenic hosts that can carry the infective stage larvae; these include shrimp, prawns, crabs, 48 frogs, water monitor lizards, centipedes, and some planarians [7. 9, 10, 11, 12]. Of planarians, 49 the predacious Platydemou...

The semi-slug, Parmarion martensi, is an intermediate host of the zoonotic nematode, Angiostrongylus cantonensis, the aetiological agent of neuroangiostrongyliasis or rat lungworm disease in humans. Rearing methods were developed for P. martensi to facilitate studies on nematode transmission and control. Parmarion martensi exhibited high survivorship when reared on a diet of dog food and fresh fruits and vegetables in temperature-controlled cabinets at 21.4°C, 98% relative humidity and 12:12 L:D cycle. Rearing containers were lined with moist paper towels for substrate and plastic pots were provided for hiding/resting and egg-laying. Under these conditions, time to first reproduction was 165.3 ± 12.3 days, fecundity was approximately 34.5 ± 7.8 eggs per adult, and hatch rate was 52.7 ± 3.2%. Survivorship post egg hatch was 86.2 ± 2.9% at 30 days (neonates had a mortality rate of about 14%) and 99% thereafter for up to a year. The demographics of laboratory-reared and wild-caught P. martensi were similar except for the weight of reproductive adults, which was significantly higher in laboratory-reared adults (4.0 ± 0.2 g) than in field-collected adults (1.5 ± 0.1 g).

BACKGROUND: Phytosanitary irradiation is used to control insect pests of quarantine concern on exported fresh horticultural products. Generic irradiation doses of 150 and 400 Gy are approved for tephritid fruit flies and all other insects, respectively. Other invertebrates such as gastropods (snails and slugs) may be classified as quarantine pests and require a disinfestation treatment. Parmarion martensi Simroth (Stylommatophora: Ariophantidae) is a semi-slug quarantine pest sometimes found on fresh sweet potatoes and other fruits and vegetables exported from Hawai'i to the continental USA. Also, P. martensi is a host of the parasitic nematode Angiostrongylus cantonensis (Rhabditida: Angiostrongylidae), the causative agent of neuroangiostrongyliasis or rat lungworm disease in humans. We conducted a study to determine if phytosanitary irradiation could control P. martensi and thereby reduce the risk of transmitting A. cantonensis in the USA. RESULTS: Two-, 12-, and 21-week-old P. martensi were treated with X-ray radiation at a dose of 150 or 400 Gy or left untreated as controls then held in the laboratory for up to 250 days. Survivorship and reproduction were recorded every 2-3 days and individual weights were measured biweekly. Irradiation at 150 and 400 Gy reduced growth and increased the mortality rate compared to untreated controls and prevented reproduction. CONCLUSION: Phytosanitary irradiation treatment at doses ≥150 Gy will prevent the establishment of viable populations of P. martensi. The literature on radiation tolerance in gastropods suggests that the internationally approved generic dose for tephritid fruit flies of 150 Gy may be effective against many slug and snail pest species.

Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis worldwide, and east Hawaii Island is a hotspot for the disease in the United States. A combination of glycoproteins with molecular weight of 31 kDa has been used as antigen to evaluate antibody response in human serum samples in Thailand with high specificity and sensitivity. In a previous pilot study, the Thailand-isolated 31-kDa proteins showed efficacy in dot-blot tests using serum samples from 435 human volunteers on Hawaii Island. However, we hypothesized that native antigen isolated from Hawaii A. cantonensis may exhibit higher specificity than the Thailand-isolated 31-kDa antigen due to potential minor variation in epitopes between isolates. In this study, 31-kDa glycoproteins were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis from adult A. cantonensis nematodes collected from rats captured on east Hawaii Island. The resultant proteins were purified by electroelution, pooled, bioanalyzed, and quantified. A subset of 148 samples from human participants of the original cohort of 435 was consented for this study, including 12 of the original 15 clinically diagnosed participants. Results of ELISA using the Hawaii-isolated 31-kDa antigen were compared with results of the same serum samples previously tested with both crude Hawaii antigen ELISA and Thailand 31-kDa antigen dot blot. This study shows a seroprevalence in the general population of East Hawaii Island of 25.0%, similar to previous findings of 23.8% seroprevalence in this cohort using crude antigen from Hawaii A. cantonensis and 26.5% using Thailand 31-kDa antigen.