Unless specified otherwise, all procedures described below were conducted in a climate-controlled room (25±3°C, 60±10% relative humidity [RH], 16-hour light: 8-hour dark [16L:8D] lighting schedule). Komatsuna ( Brassica rapa var. perviridis L. cv. Rakuten) plants (Takii & Co., Ltd., Kyoto Japan) were cultivated in soil (Ikubyou-baido: Takii & Co., Ltd., Kyoto Japan) in individual plastic pots (diameter: 90 mm, depth: 70 mm) for 4–5 weeks.

DBM were collected in a field near Kyoto, Japan, and mass-reared on potted komatsuna plants in the climate-controlled room. Eggs were collected every a few days, and hatched larvae were reared on cut plants in small cages (25 × 15 × 10 cm high).

Cotesia vestalis were obtained from parasitized DBM larvae collected in the same field. Newly-emerged adults were maintained in acryl cages (35 × 25 × 30 cm high) with 50% aqueous honey as food for 3 d to ensure mating. Female wasps were then individually transferred to glass tubes (2 cm diameter, 13 cm long) containing 50% aqueous honey as food and kept in a dark climate-controlled chamber (18±3°C, 60±10% RH, 24D) to suppress flight and prolong lifespan before use. Females were never kept for more than 6 d. At least 1 h before the start of each experiment, naive female wasps were transferred to the experimental chamber (25±3°C, 60±10% RH).

The synthetic mixture of HIPVs was prepared as follows. Pure compounds of (Z)-3-hexenyl acetate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), n-heptanal (Wako Pure Chemical Industries, Ltd., Osaka, Japan), α-pinene (Tokyo Chemical Industry Co. Ltd., Tokyo Japan, sabinene (Soda Aromatic Co. Ltd. Tokyo, Japan) and (R)-limonene (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were dissolved in triethyl citrate (TEC) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) (0.01% (w/w) TEC solution) to achieve slow volatilization. Using gas chromatography–mass spectroscopy (GCMS; Agilent 6890N/5973MSD System, Agilent, Santa Clara, CA, USA) with a TC-Wax capillary column (GL Sciences, Tokyo, Japan), the ratios of compounds in synthetic mixtures were adjusted to match those released by an infested cabbage plant ¹⁴ . The gas chromatograph oven temperature was programmed to rise from 65°C to 120°C at 3°C/min, and then from 120°C to 245°C at 5°C/min. The synthetic mixture (Blend A) was composed of sabinene, n-heptanal, α-pinene, and (Z)-3-hexenyl acetate at a ratio of 1.8:1.3:2.0:3.0 ¹⁴ . This blend was called Blend A.

We already reported that Blend A did not become more attractive to C. vestalis by adding ( R)-limonene, which was found in higher amounts in the headspaces of DBM-larvae-infested cabbage plants than intact ones ¹⁴ . Thus, to test whether C. vestalis discriminated qualitative differences in volatile blends, we added limonene to make Blend B. The ratios of sabinene, n-heptanal, α-pinene, ( Z)-3-hexenyl acetate, and ( R)-limonene were adjusted to be 1.8:1.3:2.0:3.0:1.0 by GCMS. Next, to test whether female wasps could discriminate the ratios of the four compounds in the blend (quantitative differences in the blend), we prepared a third blend (Blend C) of sabinene, n-heptanal, α-pinene, and ( Z)-3-hexenyl acetate at a ratio of 1.0:1.0:1.0:1.0. In two-choice tests, we compared either Blends A vs. B or Blends A vs. C.

Female wasps that had experienced oviposition or starvation were prepared as follows. To generate oviposition-experienced C. vestalis, we confined a second stadium DBM larva in a glass vial (2 cm in diameter, 5 cm long), and then introduced a female C. vestalis. In most cases, oviposition occurred within 5 min. Wasps that oviposited on the larvae were used for bioassays. Starvation-experienced C. vestalis were then confined in a glass vial (2 cm in diam., 5 cm long) for 2 h without food.

To prepare wasps that had been exposed to volatiles during oviposition or starvation experience, we placed a piece of filter paper (1.5 × 1.5 cm) impregnated with 10 μL of a TEC solution of a volatile blend (see below) into the tube before confining a host larva and a wasp. For control (unexposed) experiments, we used a blank piece of filter paper of the same size. To compare the effects of added limonene to those of the original blend (Blend A), we used Blend B for the oviposition and starvation experiments. In the comparison of quantitatively-different blends (Blends A and C), we used Blend A for the oviposition and starvation experiments.

Female C. vestalis were tested for their flight responses toward two potted intact komatsuna plants with a different blend. Two potted plants were placed in an acrylic cage (25 × 30 × 35 cm with three nylon-gauze–covered windows and one door) in a climate room at 25±2°C, 50–70% RH, and with continuous fluorescent light (20W, 3000 lux) without directed airflow ⁶ . We placed a piece of filter paper (2 × 2 cm) impregnated with a 0.2 g blend in a Petri dish (diameter: 3.5 cm) at the base of the potted plants. Wasps were released individually from a glass tube positioned halfway between the plants. Each repeatedly hovered over the plants inside the cage, and when it first visited a plant (landed and initiated ambulatory search), it was removed with an aspirator. The plant visited was scored as its choice. For each replicate, usually 10 wasps were tested sequentially using the same pair of potted seedlings. Each treatment had three or four independent replicates. Female C. vestalis were used only once. New plants and blends were used for each replicate.

Two-choice data were analyzed using a replicated G-test ¹⁹ . Wasps that chose neither plant were discarded from this analysis.

We first confirmed that oviposition-inexperienced (naïve) female wasps showed no significant preference between Blends A and B in the choice chamber (G-test, Gt = 5.9925, P = 0.1997; heterogeneity among samples: Gh = 3.9711, P = 0.2646; pooled effect of treatment: Gp = 2.0213, P = 0.1551) ( Figure 1a). When the female wasps had prior oviposition experience on hosts in the absence of synthetic blends, they showed an equal distribution between Blends A and B ( Gt = 1.2421, P = 0.7429; Gh = 1.2050, P = 0.5474; Gp = 0.0370, P = 0.8474) ( Figure 1b). However, when the female wasps had prior oviposition experience in the presence of Blend B, they showed a significant preference for Blend B over Blend A ( Gt = 7.9547, P = 0.0470; Gh = 2.6415, P = 0.2669; Gp = 5.3131, P = 0.0212) ( Figure 1c).

When the female wasps had experienced starvation in the absence of synthetic blends, they distributed equally between Blends A and B in the choice chamber ( Gt = 3.0677, P = 0.3813; Gh = 2.2013, P = 0.3326; Gp = 0.8664, P = 0.3520) ( Figure 1d). However, when they had been starved in the presence of Blend B, they significantly preferred Blend A over Blend B ( Gt = 9.3013, P = 0.0255; Gh = 0.6395, P = 0.7263; Gp = 8.6618, P = 0.0032) ( Figure 1e). Thus, both oviposition and starvation experience affected wasp preferences for Blend A or Blend B.

We first confirmed that naïve female wasps showed no significant preference between Blends A and C in the choice chamber ( Gt = 3.6264, P = 0.4589; Gh = 0.8118, P = 0.8466; Gp = 2.8147, P = 0.0934) ( Figure 2a). When the female wasps had previously oviposited in the absence of any synthetic blend, they distributed equally between Blends A and C in the choice chamber ( Gt = 1.7570, P = 0.6243; Gh = 0.7502, P = 0.6872; Gp = 1.0068, P = 0.3157) ( Figure 2b). Even when female wasps had oviposited in the presence of Blend A, they still showed no preference between Blends A and C in the choice chamber ( Gt = 6.0272, P = 0.1103; Gh = 5.987, P = 0.0501; Gp = 0.0400, P = 0.8415) ( Figure 2c).

When the female wasps had previously experienced starvation in the absence of any synthetic blend, they also distributed equally between Blends A and C in the choice chamber ( Gt = 1.4221, P = 0.7004; Gh = 1.3876, P = 0.4997; Gp = 0.0344, P = 0.8527) ( Figure 2d). However, when they experienced starvation in the presence of Blend A, they significantly preferred Blend C over Blend A ( Gt = 6.9032, P = 0.0750; Gh = 0.3765, P = 0.8284; Gp = 6.5268, P = 0.0106) ( Figure 2e). Thus, only starvation experience affected the choice between Blends A and C.