Nematodes’ source
The EPNs species (Steinernema feltiae (Tokat-Emir), S. carpocapsae (Tokat-Bakışlı05), Heterorhabditis bacteriophora (TOK-20), H. bacteriophora (11-KG) were obtained from the Plant Protection Department of Tokat Gaziosmanpaşa University, Turkey. Infective juveniles of the 4 species were reared on last instar larvae of the greater wax moth, Galleria mellonella (L.) described by Kaya and Stock (1997).
Mass rearing of Galleria mellonella larvae
Galleria mellonella’s larvae were reared on a artificial diet, contained 890 g flour, 222 g dry baker's yeast, 500 g glycerin, 500 g honey, 445 g milk powder and 125 g beewax in an incubator with 16/8 h lighting set at 24–25 °C (Mohamed and Coppel 1983).
Mass rearing of entomopathogenic nematode species
Ten last instar G. mellonella larvae were placed in small Petri dishes with lined Whatman paper. A suspension EPNs of infective juveniles inoculated the larvae. Petri dishes were placed in an incubator at 20–23 °C. Dead larvae was controlled frequently. Infective EPNs juveniles were obtained from infected G. mellonella larvae, using the "White trap" method (White 1927). Juveniles were kept in a refrigerator at + 10 °C.
Rearing of the Medfly
The stock culture of the Medfly, reared in the Entomology Laboratory, was used in the experiments. Fruit fly production was carried out in plexiglass adult cages with 50 mesh tulle on both sides in climate rooms with 25 ± 1 °C, 65 ± 5% relative humidity, and 16 h light and 8 h dark conditions. Eggs laid by mature individuals were left on large Petri dishes on artificial media made from a mixture of bran, wheat germ, yeast, hydrochloric acid and water. The last instar larvae were collected 6–8 days after the eggs laying to be used in the experiments.
Experiments
Experiments were conducted using 150 ml plastic containers, containing the last instar larvae and pupae of the pest on soil mixture (80% sand, 15% soil and 5% clay) sterilized at 121 °C (Chen et al. 1995). Five-to 7-day-old pupae were used in the experiments. In adult trials, EPNs were applied by placing Whatman paper on the bottom of 150 ml plastic containers and a piece of cotton soaked in sugar melted water was also left in each container. Four concentrations (0, 50, 100, 200 IJs/C. capitata stages) for each nematode species were tested against both late instar larvae, pupae and adults of C. capitata. Trials had 10 replications of each concentration and 5 individuals per repeat (50 individuals in total). After placing the 5 individuals of the pest in each plastic container, the nematodes were applied by a pipette. Only distilled water and sugar melted water were provide as food for the adults in control group. All the trials were repeated twice, on different dates, and the set up was incubated at 25 °C under 16 h dark and 8 h light climatic room conditions. Mortality rates of adults and larvae were calculated 5 days after the inoculation, while pupae were calculated 10 days later. Dead medfly larvae, pupae and adults were collected and placed in the White trap method. About one week later, EPNs emerged from insect cadavers and were examined under a stereomicroscope.
Statistical analyses
In experiments, all data were analyzed by statistical program JMP 7. Analyses were done using one-way ANOVA and used “LSMeans Student’s t test” for finding the variety in the effects of different nematode species and concentrations on Medfly.
