G. mellonella larvae
A colony of G. mellonella was reared for many years at the Center of Biological Control, Faculty of Agriculture, Cairo University, Egypt, as model insect for different traditional biological control and microbial control research studies. The larval colony was reared on the semi-synthetic diet described by Ibrahim et al. (1984) and Metwally et al. (2012). The diet consisted of two components; the first is in dry powdered form containing 20 g corn flour, 10 g milk powder, and 90 g wheat flour, and the second in fluid form of 20 ml bee honey and 20 ml glycerin. The dry components were well-mixed together, and the two fluids together. Then, the mixed fluids were added gradually to the dry mixed components with continuous mixing to reach a wetted paste that could be kept frozen till needed. The diet was placed in metal boxes (14 × 30 × 15 cm) because the wax moth larvae chew the plastic containers. A diet layer of 8–10-cm height was placed into the metal boxes, and eggs of G. mellonella were placed on a piece of paper on the diet surface. The boxes were covered by perforated metal cover top and held in the rearing room at 25–30 °C and 60–70% relative humidity. The rearing room was well equipped with ventilators to keep the temperature under 30 °C due to the raised temperature of the rearing boxes caused by the developing larvae inside. Formed pupae attached to the rearing box cover top were collected and transferred to a wooden cage of 50 × 50 × 50 cm with glass sides and a cloth sleeve door. Emerged adults were collected through the sleeve door with a small electric hover and placed in groups of ca 30 adult moths in 2-l glass containers and covered with a piece of paper secured in place by a rubber band. Females laid eggs mostly on the cover paper which was replaced daily, and the laid egg clusters attached to the paper were cut using a scissor and placed on diet in new boxes. The colony provided the needed numbers of larvae in the desired instar for the tests.
G. mellonella virus (GmMNPV)
A sudden death of larvae in some rearing boxes revealed typical symptoms of developed infection by a nuclear polyhedrosis virus. Koch’s postulates were applied and proved death of larvae by GmMNPV. Following the technique described by Parthasarathy and Rabindra (2002), the cadavers of larvae were macerated and homogenized in sterilized distilled water, and the crude was passed for primary filtration through several layers of muslin cloth. To remove the debris from the filtrate, it was centrifuged at 900 rpm for 2 min, and the supernatant was further centrifuged at 5000 rpm for 10 min. The pellet of polyhedral inclusion bodies (PIBs) was resuspended in sterile distilled water for adjusting to 2 × 108 PIBs/ml using a hemocytometer and kept in a refrigerator at 4 °C till needed for the tests.
Bioassay of GmMNPV versus G. mellonella larvae (L3)
Five concentrations from GmMNPV at 2 × 102, 2 × 103, 2 × 105, 2 × 107, and 2 × 108 PIBs/ml were prepared in sterilized distilled water. One milliliter of each concentration was mixed in 1-g diet and spread in a thin layer in glass Petri dish (5 cm in diameter). Ten larvae at the 3rd instar (L3) were placed on the spread diet and left feeding on for 24 h; thereafter, they were transferred onto untreated diet and inspected daily. The treatments were replicated 4 times using 50 larvae each. A control was set in the same manner on untreated diet. Mortality among treatments and control was recorded daily for 10 days post-treatment. The test larvae originated from a new colony reared from disinfected eggs of G. mellonella on the same diet in disinfected metal containers to ensure that they were healthy and not infected with the virus.
Larvae of G. mellonella fed on diet treated with GmMNPV (2 × 108 PIBs/ml in 1 g diet) were picked up at the 5th and 7th day post-treatment and fixed in Buin’s fixative. Fixed larvae were rinsed in 70% ethyl alcohol for 6 h and dehydrated in ascending ethyl alcohol concentrations of 80, 90, 95, and 100% each for 5 h, followed by clearing in xylene for 12 h and transferred in xylene: paraffin wax 2:1, 1:1, and two times each for 12 h in pure paraffin wax at 40 °C. The wax-impregnated larvae were poured in blocks over iced water and trimmed for cross serial sectioning by microtome at 6–8 microns. The serial sections were glued on glass slides by Mayer’ Albumin and left on a hot plate (30 °C) for adhering. The wax was removed by washing two times each for 20 min in xylene. The sections adhered to the slides were hydrated in descending ethyl alcohol concentrations of 100, 95, 90, 80, and 70% each for 15 min and stained either with eosin or hematoxylin and then, dehydrated in ascending ethyl alcohol from 70 to 100% and cleared in xylene before mounting in Canada Balsam and dried at 40 °C for 3 days. The prepared cross sections were examined by light microscope connected to a computer for photography to show the polyhedral inclusion bodies of the virus in cells of different tissues and disintegrated cell contents in the body cavity of the infected larvae.
Control of G. mellonella on stored wax foundations by GmMNPV
The possibility for controlling larvae of the wax moth using GmMNPV (2 × 107 PIBs/ml) on stored wax foundations was tested. Twenty sheets of beeswax foundations previously sterilized by formalin vapor were sprayed by the virus concentration using a hand atomizer each on both sides and left standing vertically for evaporation of the sprayed water leaving the PIBs on the wax behind. Thereafter, they were placed horizontally, and each received ca 200 eggs of G. mellonella and left for 6 days to enable the newly hatched larvae to start feeding and settle themselves on the wax sheets. The treated wax sheets were stored vertically in a wooden box at room temperature of 25–30 °C and 60–70% RH. Another surface-sterilized 20 wax sheets were sprayed only with distilled water and received ca 200 eggs of G. mellonella served as control. The infested wax sheets harboring the larvae were planned to store for 12 months and were weighted together monthly, and the loss percent was calculated. A blank control of the healthy larvae without treatment was set up, and the wax foundations were kept in similar wooden box under the same storing conditions.