Samples collection
Samples of terrestrial snails and slugs were collected from infested plants by hand picking from Cairo, Egypt, during 2016-2019. Snails and slugs were identified according to Godan (1983); Cowie (2002); Auffenberg and Stange (2009); Baker Skinner Park (2009); and Azzam and Tawfik (2011).
Rearing of gastropods
After collecting and identifying the gastropod samples, they were washed thoroughly using a fine metallic net under strong stream of tap water and then washed again by distilled water. Some individuals were placed in a Petri-dish with distilled water and examined under a research microscope, to check for the presence of the microorganisms. This process was repeated several times until the complete removal of all external associated organisms.
Rearing of Eobania vermiculata (Müller) snail and Limax flavus L. slug were carried out by placing the examined individuals in plastic cages, sterilized with ethanol alcohol, substrated with sterilized clay, and irrigated by distilled water. After egg laying, the eggs were washed gently by distilled water and transferred to sterilized Petri dishes contained sterilized clay.
The Petri dishes were then placed in a dark place until hatching. The newly hatched gastropods were transferred gently to new sterilized cages as mentioned before and supplied with some lettuce leaves after washing them thoroughly by a strong stream of tap water to remove associated organisms then washed again by distilled water and introduced to the newly hatching gastropods.
Isolation of nematodes from eggs
The collected snail eggs were gently washed by tape water then distilled water and examined under a microscope to remove any external associated nematodes or any microorganisms. If microscopic examination showed any organisms association, the egg washing should be repeated again and again until eggs are looked totally free from these organisms. After the complete removal of external associated microorganisms, the eggs were screened for parasitic nematodes by placing them individually in small Petri-dishes, 6 cm in diameter, with little distilled water (1 ml). The dishes were examined daily by a microscope for releasing nematodes and the distilled water was recompleted. Every 3 days, two eggs were dissected to check the presence of the parasitic nematodes inside. The isolated nematodes were identified according to Andrassy (1976), Azzam (2003), Sudhaus (2018), Tandingan De Ley et al. (2014, 2016), and Singh et al. (2019).
Count of nematodes
Counting of the released nematodes of recovering individuals, to be used in the infection of tested species and stages, was carried out by taking a 5 μl of the nematode suspension (by means of a micropipette) after shaking it thoroughly, it was placed on micrometer slide, examined microscopically, and counted the number of nematodes. This procedure was repeated 4 times. The mean number was calculated and multiplied by 200 to estimate the number of nematodes in 1 ml. To prepare adjusting concentration, some of this solution may be diluted to the concentration needed.
Infection of snails and eggs
The isolated nematodes infectivity was investigated toward lab bred of E. vermiculate snails (Fig. 1), L. flavus slug (Fig. 2), and non-local giant terrestrial snails, Achatina fulica Bowdich (Fig. 3) which have not been found in Egypt (one snail obtained from the Agricultural Quarantine Office). This sample deposited 125 eggs in the laboratory during examination and identification. These eggs were treated as mentioned above and exposed to infection by the isolated nematodes.
The nematodes were also tested for the infection of eggs of the abovementioned three species (Figs. 4, 5, and 6) respectively at 22±2 °C, using the same technic previously described by Azzam (1998).
The relative infectivity of the isolated nematodes to the abovementioned gastropods was determined by placing the 20 lab bred individuals of each species and stages individually in a Petri dish (9.5 cm in diameter for snails, slugs, and eggs). A little amount of distilled water contained isolated nematodes (1.5 ml contained 500 I.J. S. for snail and slugs and 0.5 ml contained 100 I.J. S. for eggs) were placed in their relevant Petri dishes. Another 10 individuals from each species were treated similarly to abovementioned groups but using distilled water only without adding nematodes as control groups.
For the infection of the individual adult of the non-local giant terrestrial snail, A. fulica (Fig. 3), 2500 I.J.S. in 3 ml was added into a Petri dish (14 cm in diameter). The concentration increased than those used for adults of E. vermiculata and L. flavus, to be adequate for its large size. The size of the Petri dish and the volume of nematode suspension were also increased for the same reason. For the comparison of the suitability of this parasitic nematode to infect different hosts, the following capacity index was calculated for each host guided by Azzam (1998).
$$ \left(\mathrm{C}.\mathrm{I}=\mathrm{M}\times \mathrm{R}/\mathrm{T}\right) $$
where M is the mortality rate of pests at the end of 6 weeks, R is the percentage recovery of the infected stage of nematodes in infected individuals, and T is the time needed for the nematodes to reach the infective stage after initial infection.
Statistical analysis
The time needed for the nematodes to reach infective stage after initial infection, body length of nematode in different stages, ratios of mortality, and individuals recovered nematodes were analyzed by ANOVA and T test value using PSPP program.