Rearing of Galleria mellonella (L.)
Larvae of G. mellonella were collected from infested bee hives, reared in jars (2-kg capacity) until emergence of the moths. Their laid eggs were collected. The rearing was carried out in accordance with the methodology adapted by (Dutky et al. 1964), using an artificial diet modified by (Huang et al. 2010) under the laboratory conditions of 28 ± °C, 60% R.H., and 16:8 L: D photoperiod.
Rearing of the parasitoid, Bracon hebetor Say.
A stock culture of B. hebetor was obtained from infested flour with the Mediterranean flour moth, Ephestia kuehniella (Sulz.), collected from warehouses of flour mills, Giza Governorate, Egypt. Infested flour was placed in plastic containers, until emergence of parasitoid’s adults. Afterwards, newly emerged females and males were isolated in a separated container, supplied with a drop of honey as food source, and the last larval instar of G. mellonella for laying egg to start the parasitoid culture. The containers were kept in an incubator at 28 ± 1 °C, 60% R.H. (Farag et al. 2015).
Biological studies were carried out under the laboratory conditions of (28 ± 1 °C, 60–70% R.H. and 16:8 L: D photoperiod).
Durations of immature stages (egg-pupa)
Newly emerged adults of B. hebetor were paired (female and male) provided with 10 larvae of 5th larval instar of G. mellonella, for 24 h, in a small plastic container (10 cm in height, 7 cm in diameter), containing droplets of honey, covered with white muslin kept in position by rubber bands. The paralyzed larvae were separated individually in Petri dishes. Only one wasp egg was left on each host. The paralyzed larvae were investigated daily, using a stereomicroscope, to record the durations of immature stages from egg to pupa (30 replicates were used).
Newly emerged parasitoid adults were placed individually in small glass vials and fed on droplets of honey until their death. Longevity of each sex was recorded (30 pairs; males and females).
Newly emerged females of B. hebetor were placed with males for 24 h in a 500-ml plastic jar for mating. The opportunity for mating was provided as 80% of virgin B. hebetor females mate within the first 15 min of being in the presence of male (Ode et al. 1995; Dabhi 2011). After 24 h, B. hebetor females were separated from the males and introduced individually in a small plastic jar (10 cm in height, 7 cm in diameter), containing 10 of 2nd and last larval instars of G. mellonella, each age individually, covered with white muslin and kept in position by rubber bands. After 24 h, females were carefully transferred to a new small plastic container containing fresh larvae of a given host age. This procedure was repeated until the death of female parasitoids. Number of larvae paralyzed and number of eggs on the paralyzed larvae were counted daily, using a stereomicroscope (30 replicates). Total number of laid eggs, periods of pre-oviposition, oviposition, and post-oviposition were recorded.
The experiments were carried out at the apiary of the Bee Research Department, Plant Protection Research Institute, Agriculture Research Center, Giza, Egypt, under the environmental conditions from September to February the winter season of 2018–2019.
Efficacy of releasing B. hebetor against G. mellonella in honey bee colonies
Honey bee colonies
Six Carniolan (F1) of A. mellifera carnica honey bee colonies, provided with mated hybrid queens, each at the same age, were prepared in a control experiment at the apiary. Each colony, contained 1.125 kg of adult bees (about 12,300 bees), 2 of sealed and unsealed brood combs, 2 combs of honey and pollen, and one empty fully drawn comb.
The colonies were randomly divided into 2 groups 3 honeybee colonies each. The first group (treated group) was provided by 100 of 5th larval instar of G. mellonella and 30 female parasitoids of B. hebetor released weekly, starting from September 2018 to February 2019 for each colony. After that, colonies were closed for 6 h to avoid way out of the parasitoid attracted by the light. The second group (untreated group) was provided weekly only by 100 of 5th instar larvae of G. mellonella for each colony and used as a control.
Efficacy of releasing B. hebetor against G. mellonella in stored wax combs
The experiment was carried out at an apiary store, using 6 langstroth hives, each had 10 wax combs. The hives were randomly divided into 2 groups; the first was provided weekly by 30 females of B. hebetor hive, while the second was provided by formic acid 60% hive and used as control. The experiment started in the 1st week of September 2018 until the end of February 2019.
The following parameters were assayed to evaluate the efficacy of releasing the parasitoid B. hebetor in the honeybee colonies and in the stored wax combs experiments:
Number of dead and/or survived larvae of G. mellonella/colony.
Infested areas of G. mellonella larvae (inch2)/colony measured by using a frame divided into square inches.
Number of infested tunnels/colony.
Data were analyzed in a randomized complete block design (ANOVA) by MSTAT-C version 1.41 (Sendecor and Cochran 1980), and using the graph pad PRISMA version 3.03 for windows, software. All means were compared by Duncan’s multiple range test at level 0.05 (Duncan 1955).