Trichogramma brassicae (strain Y175) wasps originated from a cabbage field in The Netherlands and were reared on eggs of Pieris brassicae for several generations after collection. Only mated, 2 - 5 day-old naïve female wasps (no contact to eggs prior to the experiment) were used for the experiments, with the exception of the flight chamber test where oviposition-experienced females were used.
Pieris brassicae adults were obtained from a laboratory colony maintained on Brussels sprout plants (Brassica oleraceae L. var. gemmifera cv. Cyrus). Mated females and males were obtained by taking copulating pairs from the rearing. Virgin females and males were separated in the pupal stage based on the last segments of the abdomen.
Response to butterfly odours
The experiments were carried out in a two-chamber-static-air-flow olfactometer (see figure 1). The olfactometer consisted of a cylinder made of acrylic glass (18 cm high, 12 cm ) divided into two chambers by a vertical plate. No airflow was generated. On the top of the cylinder, a removable walking arena (2 cm high, 9 cm ) was placed consisting of plastic gauze (mesh 0.1 mm) with a plastic rim and covered with a glass plate. The experiments were carried out in the laboratory at 23 ± 1°C using a fibre optic light source (Euromex coldlight illuminator EK-I, The Netherlands) above the olfactometer. Two adult butterflies per chamber were introduced as the odour source. The time spent by the wasps in one of the two odour fields was observed for 300 seconds. Each day 10-15 naïve wasps were tested, for a total of 40 wasps per combination. To avoid biased results due to possible side preferences of the parasitoids, the olfactometer was rotated 180 after every third wasp tested.
Response to butterfly anti-aphrodisiac
In the same two-chamber olfactometer, the response of naïve T. brassicae wasps towards the anti-aphrodisiac (benzyl cyanide) of mated P. brassicae females was tested. A virgin butterfly female was painted with 10 l of a benzyl cyanide (Aldrich, purity 99%) solution in hexane and tested against a virgin female treated with 10 l of hexane. Five different doses of benzyl cyanide were tested: a) 10 g, b) 2 g, c) 1 g, d) 0.2 g, and e) 0.1 g. After each 5th wasp, the test and control butterflies were replaced. A total of 40 wasps was tested per dose.
Mounting on butterfly
Mounting behaviour of T. brassicae wasps was tested in a two-choice bioassay conducted in a plastic container (9 cm high, 13.5 cm diameter, compare fig. 1). Two adults of P. brassicae were placed in the arena after cooling down in a refrigerator (± 4 ºC) to decrease mobility. A naïve T. brassicae wasp was introduced and continuously observed until it climbed onto one of the two butterflies. The body part on which the wasp mounted was recorded. When a wasp did not mount one of the two butterflies within 5 minutes, a “no response” was recorded. After each 10th wasp, the butterflies were replaced. For each combination, 40 wasps were tested.
An additional mounting experiment tested two virgin butterfly females, one painted with 10 l of the anti-aphrodisiac benzyl cyanide dissolved in hexane (2 g) and the other painted with 10 l hexane. The experimental conditions were the same as described above, except for the following: both butterflies were placed under a small gauze mesh (3 x 6 cm, 2 mm mesh size) to prevent them from moving and to exclude visual cues. A naïve wasp was introduced and continuously observed until it went into a cage and mounted the butterfly. Butterflies were treated with 10 l of benzyl cyanide solution and hexane, respectively, every 10 minutes. Ten wasps were tested on each pair of virgin butterflies and a total of 40 wasps were tested.
Phoresy on butterflies
Experiments were conducted in a flight chamber installed in a greenhouse compartment under the following conditions: 25±3°C, 50-80 % RH. In a glasshouse compartment (4 x 2.5 x 3.5 m), a tent (3.3 x 1.8 x 3 m) made of white sheets was constructed. No directed airflow was used, but a turbulent airflow inside the tent was present as the greenhouse compartment was continuously ventilated. Observations were made under natural daylight. A Brussels sprouts plant, 8-12 weeks old, was placed on one end of the table. A mated butterfly female with a high egg load was offered to a wasp in a small arena on the other end of the table approximately 1.5 meter away. Both the butterfly and the wasp were observed and their behaviour recorded using The Observer software 3.0 (Noldus Information Technology 1993©). When a Pieris female carrying a T. brassicae wasp did not land on the cabbage plant after take-off, it was scored as a non-responder. When a Pieris female landed on the plant and started to oviposit, the position and movement of the wasp was scored using the following descriptors: a) lost (wasp not found back on the butterfly), b) reaches the host habitat (wasp was found on butterfly but lost trying to descend onto the plant), c) reaches host plant (wasp climbed onto the plant but was lost thereafter), and d) observed parasitism (wasp found butterfly eggs and parasitized them during the observation).