The greater wax moth (G. mellonella L.) larvae were obtained from infested hives and reared in jars (2 kg capacity) until the emergence of moths according to the technique described by Birah et al. (2008). Similar constituents, but with different proportions, were tried later by Huang et al. (2010). Fully grown larvae of G. mellonella were also used for isolation and production of entomopathogenic nematodes.
Rearing of Spodoptera littoralis
A field strain of S. littoralis was obtained from an open field of the vegetable farm at Giza Governorate, Egypt, transferred to the laboratory, and reared at 25 °C ± 2 °C and 65–75 RH%. S. littoralis larvae were placed in glass jars and fed on castor leaves (Ricinus communis L.) (Zhang et al. 2019a). The jars were provided daily with castor leaves as a source of food for the larvae. The pupae were transferred to suitable cages for mating; the emerging adults were fed on 20% honey solution even lay eggs (Ibrahim 1974).
Rearing of Agrotis ipsilon
The A. ipsilon larvae used in this study were collected from an open field of a vegetable farm at Giza Governorate, Egypt. The 4th instar larvae were reared singly inside plastic tubes (1.5 cm in diameter, 15 cm in height) or in small groups in plastic jars to avoid cannibalism until the last instar of the larvae developed to the pupal stage. All pupae were transferred into suitable cages for mating, the emerging adult moths were fed by a on 20% honey solution till laying eggs. All of the rearing procedures for A. ipsilon were carried out at 25 ± 1 °C and 75 ± 5% RH (Zhang et al., 2019b). The newly hatched larvae were transferred into small plastic jars and provided daily with castor leaves as a source of food.
Soil samples were collected from Nubarya Elbhaira governorate, Egypt, in the years 2018 and 2019 every 15 days under citrus trees (Citrus sinensis and Citrus tangerine), under guava trees (Psidium guajava), and under Egyptian clover (Trifolium alexandrinum). Sandy soil was chosen for the soil samples’ collection. About 1 kg of soil/sample was collected, 10–15 cm below the soil surface. Samples were kept in plastic sacs labeled with the necessary information. The soil sacs were then placed in an icebox and transferred to the laboratory. In the laboratory, soil samples were placed in cups and fully grown larvae of (G. mellonella) were added onto the soil samples.
G. mellonella baiting technique modified after Bedding and Akhurst (1975). Soil sample of each sac was thoroughly mixed and then divided into 10 plastic cups (about 100 ml volume). Five G. mellonella late instar larvae were placed onto the soil surface/cup, and the cups were covered by their lids, turned upside–down every 24 hrs to mix in the soil and kept at the temperature of 25 ± 2 °C. The cups were examined daily and throughout 6–10 days later, dead larvae, suspected to be nematode–infected, were picked up carefully, rinsed several times in distilled water, and incubated at 25 °C in extraction White traps. Each White trap contained only one dead larva, using the method originally described by Dutky et al. (1964). Infective juveniles of nematode were harvested daily from the White traps by receiving them in 0.1% formaldehyde solution and stored at 15 °C (White 1927). Effectiveness of the infective juveniles of nematode was tested against larvae and pupae.
Infectivity of the two strains of Heterorhabditis sp. to 3rd instar larvae and 1-day-old pupae at 25 °C ± 2 of the S. littoralis and A. ipsilon was tested. Ten 3rd instar larvae or 10 1-day-old pupae were placed at 1 cm depth from the surface and treated with each of the tested nematodes strain. The infection took place in plastic cups (100 cc capacity), filled with 1 cc sterile sandy soil, and covered with plastic lids. The nematode suspension was poured in the vials and mixed with the soil at 5 concentrations 500, 1000, 2000, 4000, and 8000 IJs/cup. Five replicates were used for each concentration. The numbers of dead larvae or pupae were recorded after 6 days of treatment. Water content in the soil was always kept at 20% of the soil weight. The control treatment was carried out using distilled water.
Production of entomopathogenic nematode
Ten larvae were used for each concentration. Full-grown larvae of G. mellonella were confined, individually, in plastic cups (100 cc capacity) lined with filter paper and covered with plastic lids. The infection took place using five concentrations of 1, 2, 4, 8, and 16 IJs/cup for each of the 2 strains of Heterorhabditis sp. Infective juveniles were harvested daily using White traps according to White (1927). The whole number of IJs produced/larvae was estimated. All experiments were carried out in a conditioned laboratory at 26–27 °C and 50–60% R.H.
Mortality rates were corrected according to Abbott’s formula (Abbott 1925); the toxicity lines and LC50 values were calculated according to Finney (1971). The mass production of the two strains was statistically analyzed by ANOVA, and the mean values were separated by the least significant difference (L.S.D.) procedure (Snedecor and Cochran, 1980). Also, t test between the average production of the two strains at different concentrations or at each one was calculated.