3 EXPOSURE PROCEDURE

FOR TEST purposes the various developmental stages were counted into batches of 50 insects and placed on the bottom of a small petri dish lined with black filter paper. To prevent the possible escape of any surviving insects, the petri dishes were subsequently transferred to wide-mouth glass jars (fitted with filter paper lids), which had been cooled to −20�C inside a walk-in freezer unit. All developmental stages were then exposed to −20�C for periods ranging from 1 minute to 4 hours. After freezing, 10 g of finely sieved culture medium was added to each jar which was then incubated at 25�C and 70% RH. After 24 hours the jars containing the larvae and adults were resieved, and the number of survivors counted. The jars containing the eggs and pupae were resieved after two weeks. The number of eggs that had hatched and the number of pupae that had emerged as adults were then determined.

Parallel tests with an equivalent number of untreated insects acting as controls were conducted for each developmental stage. These were incubated at 25�C and 70% RH. The tests on eggs, larvae, pupae, and adults were conducted without replication because of the difficulty of obtaining large numbers of these stages at any one time. Treatments were repeated three times for each developmental stage to confirm the results. The percent mortality for each developmental stage was calculated using the formula (x−y)/x (100), where x is the percentage survival in the control and y that in the frozen sample.