EPNs, T. castaneum adults, the great wax moth, Galleria mellonella L. cultures constituted the main materials of the study. Experiments carried out under laboratory conditions in the Nematology Laboratory of the Central Research Institute of Plant Protection (Ministry of Agriculture and Forestry of the Republic of Turkey) in 2020.
Infective juveniles of Steinernema carpocapsae (Tokat, Bakışlı 05), S. feltiae (Tokat, Emir), Heterorhabditis bacteriophora (TOK-20), H. bacteriophora (11-KG) were obtained from Tokat Gaziosmanpaşa University. All nematode species were reared on G. mellonella mature larvae as described by Kaya and Stock (1997).
Rearing Galleria mellonella larvae
A special nutrient diet, containing 890 g of flour, 222 g of dry baker's yeast, 500 g of glycerin, 500 g of honey, 445 g of milk powder and 445 g Flour, bran, milk powder and yeast, mixed and then poured on a mixture of honey and glycerin, was prepared (Mohammed and Coppel, 1983). Galleria mellonella eggs were placed on a food medium in 1 lt glass jars and then placed in the incubator (16/8 h lighting set at 23–24 °C) for hatching and development of the larvae.
Rearing of entomopathogenic nematode species
Mature instar larvae of G. mellonella were used for rearing the EPNs. The larvae were placed on Whatman paper (White, 1927), soaked in sterile water, in small Petri dishes (diameter of 6 cm). The 2nd and 3rd stages of infective nematode were collected from the water by a dropper and applied on the G. mellonella larvae. Then, the lids of the Petri dishes were wrapped by a parafilm and placed in the incubator (20–23 °C). The Petri dishes were inspected every 10 days. The obtained juveniles were kept in a refrigerator (10 ºC).
Rearing of T. castaneum
Tribolium population grown in the Stored Crop Pests Unit of the Entomology Department, Ankara Plant Protection Central Research Institute, Turkey was used in the study. A mixture of dry yeast and wheat flour (1: 3) was used in the cultivation of Tribolium. 250 g of the nutrient mixture, sterilized at −18 °C for 120 h, was placed in a 1 L glass jar with a perforated lid for ventilation. Glass jars were kept in climate chambers at 25 ± 1 °C and 60 ± 5% RH. 500–750 adults were placed in each jar for oviposition and after a week, adults were removed from by passing through a 30 mesh sieve. The screened adult individuals were placed to a new jar containing food, and the continuity of the cultures was ensured.
Laboratory experiments of EPNs
For each nematode species, the experiments were carried out in plastic Petri dishes (9 cm) under the same conditions 3 times on different dates with 10 replicates per repeat and at 2 different temperatures (25 and 15 °C) in a climate chamber. T. castaneum adults (10 individuals) were placed by the help of soft forceps into Petri dishes with 5 g sterilized wheat crumbs bedding. Then, the EPN isolates prepared, using distilled water at 250, 500 and 1000 IJs/ml, were applied directly into the Petri dish. After the application, the Petri dishes were covered by a parafilm. Only pure water was used in the control group. The vitality of the adult individuals in the Petri dishes was counted regularly at the end of 48, 72 and 120 h and the mortality % were calculated. Cadavers were taken place "White trap" and were examined under a stereomicroscope. After one week, EPNs juveniles were obtained from infected T. castaneum adults.
Duncan multiple comparison test was performed with SPSS statistics 17. Square root transformation was applied to non-normally distributed data, followed by ANOVA (Duncan test).