Box 2.

Female orientation to male pheromone.

For the purpose of evaluating the response of females towards the male pheromone, an indicator of intra-specific recognition in lek-forming tephritids, field cages are set up with two potted trees inside, which are virtually divided into two sectors. Each sector contains one tree. The test involves two steps. In the first, it is determined whether females orient to the pheromone of con-specific males and 25 mature virgin females of a given population are released into the field cage during the period of sexual activity. Fifteen minutes later, 3 “artificial leks” consisting of cylindrical metal wire-mesh containers (3 cm diam., 7 cm long) with 7 sexually mature males inside (Figure 1) are hung in one of the trees while 3 containers without males are hung in the other tree. Once the females and the containers are placed in the cage, an observer scores the number of females in each sector, in each tree and those on the artificial lek. These parameters are recorded every 15–20 minutes, with at least six observations made during the period of mating activity in each cage. If significant differences in preference towards male-containing leks are found (see below), it is possible to continue with the second step, which involves the evaluation of the ability of the females to distinguish between male pheromones of different populations, morphotypes or species. To do so, 25 mature virgin females from two different populations are released inside each field cage, into which artificial leks with males from the same two populations are hung from the trees. Each tree houses 3 containers from one population. In order to identify females from each origin, the day before the test flies are painted with a dot of water based acrylic in their thorax. Colors are randomly assigned and permuted every day. Observations are performed as described previously. To prevent pheromone contamination, trees and cages are washed with pressurized tap water at the end of each test. The containers used for the artificial leks are also washed with hot water and dried in an oven at 150 °C for one hour. To avoid females behaving differently due to adaptation of sensory organs and due to habituation in the brain, males and females should be kept in separate rooms so that females have no previous experience with the male pheromones. Data from the daily observations within each cage (i.e. replicate) are added to obtain an overall measure of the location of the females throughout the experiment. Statistical analysis is done by means of a paired t-test or the corresponding non-parametric Wilcoxon paired test as appropriate, in which the number of flies registered in the area, in the tree and at the artificial lek is compared between sectors. In the first step (i.e. field cages with empty control containers), significant differences indicate that females use chemical cues to find males. In the second step (i.e. comparing male types), significant differences indicate that females orient towards one male type, while non-significant differences indicate either lack of capacity to discriminate or no preference towards any male type.