Because lettuce is a winter crop in South Florida, we were able to conduct the experiments from September through April in 2013 and the same in 2014. Experiments were conducted in greenhouse only because there is not a method to infest L. trifolii in field settings. Relying on natural infestations was not possible as insecticides are constantly applied in fields nearby. Plants were grown in greenhouse for about seven weeks and then moved to the insectary room maintained at 27 °C and a photoperiod of 14:10 (L:D) h. Plants were watered and fertilized adequately while kept for two days in the insectary room for L. trifolii testing. Plants were transferred to a greenhouse after the L. trifolii test and allowed to grow until seed maturity.

Fully expanded leaves from the middle to upper portion of the plants (i.e. leaves in positions 5 to 9 numbered starting from the first true leaf at the bottom of the plants) were selected for L. trifolii greenhouse experiments2 because L. trifolii prefers these leaves over both the older and younger leaves. A no-choice test was performed to assess the reaction of plants to insect feeding. A clip cage (radius = 1.9 cm, height = 2.8 cm) was attached to a leaf of an individual plant supported by a bamboo stick. Due to the difficulties in determining the sex of L. trifolii accurately and quickly, we selected mating pairs to ensure the number of female flies at least 50% in each clip cage. Two mating pairs of L. trifolii less than 24 h old were aspirated into a glass tube and released into each clip cage. The flies were allowed to feed on a leaf for 48 h.

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