2.5. Male response experiments

To test geographic variation in male attraction to pheromone lures in different regions, two field trapping experiments, each with two to four replicates were conducted at each of the three sites in a complete randomized block design (Table ​(Table2).2). In experiment 1, the attraction of five synthetic pheromone blends was compared, which were prepared at Pherobank BV (Wijk bij Duurstede, the Netherlands). Each pheromone lure consisted of a red rubber septum that was loaded with 100 μL of hexane containing 5 mg of the major component Z11‐16:Ald (100%), while the other compounds were loaded in amounts relative to Z11‐16:Ald (see Table ​Table2).2). In experiment 2, the effect of the antagonist pheromone compound Z11‐16:OAc on H. armigera male attraction was tested. We focused on this antagonistic compound because it is part of the sex pheromone blend of the closely related species H. assulta that occurs sympatrically with H. armigera in China.51 For experiment 2, we prepared two synthetic pheromone lures in the laboratory. Each pheromone lure consisted of a red rubber septum that was loaded with 100 μL of hexane containing 300 μg of Z11‐16:Ald (100%) and other compounds relative to 300 μg of Z11‐16:Ald, so that one treatment consisted of 100% Z11‐16:Ald and 5% Z9‐16:Ald (which are the two critical sex pheromone components of H. armigera and to which we refer as the H.a blend), and another treatment consisted of 100% Z11‐16:Ald and 5% Z9‐16:Ald + 10% Z11‐16:OAc (H.a + Z11‐16:OAc blend) (Table ​(Table22).

Compositions of pheromone lures used in the fields

In experiment 1, compound concentrations were as follows: 100% = 5 mg, 2.5% = 0.125 mg, 1.4% = 0.07 mg, 4% = 0.2 mg, 6% = 0.3 mg, 0.6% = 0.03 mg, 0.3% = 0.015 mg.

In experiment 2, compound concentrations were as follows: 100% = 300 μg, 5% = 15 μg, 10% = 30 μg.

All lures were hung in bucket traps (Pherobank BV) attached to a wooden pole and positioned at a height of approximately 1.5 m above the ground, and distributed at least 30 m apart in the field. The males caught in the traps were collected and counted every day. All the lure experiments were conducted in the field in each region (Table ​(Table1).1). For experiment 1, at all field sites two rubber septum lures per treatment at the same time were used and the treatments were rotated daily over five nights (ten replicates per treatment) to minimize possible position and odorant effects. For experiment 2, in Spain, four rubber septum lures per treatment at the same time were used and the treatments were rotated daily over two nights (eight replicates per treatment). In Australia, two rubber septum lures per treatment at the same time were used and rotated daily over five nights (ten replicates per treatment). In China, three rubber septum lures per treatment at the same time were used and rotated daily over three nights (nine replicates per treatment).