Development of a recombinase polymerase amplification (RPA-EXO) and lateral flow assay (RPA-LFA) based on the ITS1 gene for the detection of Angiostrongylus cantonensis in gastropod intermediate hosts.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai'i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies µL-1 to ~1 copy µL-1. All three assays consistently detected 50-100 copies µL-1 in triplicate and qPCR was able to detect ~13 copies µL-1 in triplicate. RPA-EXO was able to detect 25 copies µL-1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies µL-1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Reduced Emergence of Cylas formicarius elegantulus (Coleoptera: Curculionidae) from Sweet Potato Roots by Heterorhabditis indica.

Okinawan sweet potato, Ipomoea batatas, is an important food staple and export crop for the Island of Hawaii. Cylas formicarius elegantulus, sweet potato weevil, is a major quarantine pest that causes severe destruction to the crop. Root malformation and a bitter taste occur when larvae feed and tunnel within the storage root. Off-grade roots are often left in the field after harvest and serve as a reservoir for the weevils. Current management involves the unsustainable practice of moving to virgin land for the next cropping cycle. Strains of Heterorhabditis indica isolated from the Hawaiian Islands were tested for their efficacy at causing mortality of C. formicarius and reducing the emergence of adults from infested roots. In well plate assays, H. indica caused mortality of 88% larvae, 96% pupae, and 4% adults after 48 h. When applied to infested roots, the nematodes caused an average mortality of 78% larvae, 66% pupae, and 32% adults. Greater mortality was observed at the highest inoculum levels (10,000 infective juveniles per storage tuber) but a reduction of 90% inoculum density was still effective at weevil management. In simulated field trials, infestation of storage roots was reduced by 42-99.6% when planted among infested roots that had been inoculated with H. indica. Rates of 2.5 billion IJs/hectare were just as effective as 5 billion IJs/hectare. Application of local H. indica strains in sweet potato production has the potential to manage C. formicarius populations and allow for consecutive cropping seasons.

Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus).

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Short-Term Physiological Response of a Native Hawaiian Plant, Hibiscus arnottianus, to Injury by the Exotic Leafhopper, Sophonia orientalis (Hemiptera: Cicadellidae).

Sophonia orientalis (Matsumura), also known as the two-spotted leafhopper, is a widespread exotic pest of many economically important crop plants and ornamental plants in Hawaii. Sophonia orientalis is highly polyphagous and is a major threat to some of the native endemic plants. Despite the successful establishment in Hawaii, interactions of S. orientalis with its host plants remain poorly understood. Previous studies primarily focused on distribution, parasitism, and oviposition of S. orientalis in Hawaii, whereas plant physiological responses to the leafhopper's injury, and, specifically, gas exchange rates in plants, have not yet been described. In this study, we examined a short-term physiological response of a native Hawaiian plant, Hibiscus arnottianus (A. Gray), to injury by S. orientalis. We also explored whether Camellia sinensis (L.) Kuntze, a native host plant of S. orientalis in Asia, exhibits a similar response. We found that H. arnottianus plants demonstrated a rapid (2-d) physiological response to injury accompanied by 40% reduction in rate of photosynthesis and 42% reduction in rate of transpiration, whereas C. sinensis did not exhibit any reduction in gas exchange rates. We did not record any changes in plant chlorophyll levels after plant injury in either species. Our results suggest that H. arnottianus is responding to the leafhopper feeding with a generalized wound response predicted for novel plant-insect herbivore associations. We discuss potential future directions for studies which might focus on host plant responses to S. orientalis in its native versus introduced range.