2.4. Investigation of Angiostrongylus larvae from snail intermediate hosts

Aquatic snails were collected by the local health volunteers, as per traditional snail collection methods from natural water sources in the village. The method was similar to that used by local villagers to gather snails for food consumption. GPS coordinates were recorded at the points where snails were collected.

Snails were examined and identified to species level based on morphological characteristics following Brandt (1974) and Ng et al. (2020). Snails were euthanized at 0 ^๐C for 20 min and had their shells removed. The foot and mantle parts of each snail were removed and cut into smaller pieces and incubated in artificial tissue digesting solution: 1% HCl-1% Pepsin (1:10,000; equivalent to 10,000 FCC units/mg; VWR International, UK) at 37 ^๐C for an hour. The digestive suspension was stirred every 15 min during the digestion period. Tissue debris and suspension were filtered through an aluminum sieve (No. 25, mesh size 700 um) and left to sediment for at least 20 min in a small glass bowl (10 cm diameter). Approximately two-thirds of the supernatant was then decanted, and the remaining sediment was microscopically examined for the presence of L3 Angiostrongylus spp. Third stage Angiostrongylus larvae (L3) were morphologically identified using the following characteristics: the presence of subterminal caudal constriction (pointed tail tip), a pair of cephalic chitinous rods, and body length of 480–520 μm (Lim and Ramachandran, 1979; Moreira et al., 2013). The Angiostrongylus larvae were counted and pooled per snail and preserved in 70% ethanol at −20 °C for subsequent molecular identification. The rate of parasitic infection, mean abundance (mean number of parasites found in all hosts in the population), mean intensity (mean number of parasites found in individual infected hosts), and the range of L3 Angiostrongylus infection in the snails were estimated using Quantitative Parasitology version 3.0 (Reiczigel et al., 2019).