*2.2. Experimental Treatments*

In total, we had 242 females and 171 males. The insects were randomly allocated to each treatment in which beetles were injected ventrally either 5 or 10 μg of JH type III (Sigma, St Quentin Fallavier, France) in 5 μL of Ringer: acetone (9:1) solution between the 2nd and 3rd sternite region using a 10 μL Hamilton syringe (30 G) (Hamilton Company, Switzerland). Control males and females received 5 μL of Ringer: acetone solution only, but otherwise, both groups were treated identically. Before injection, the beetles were anesthetized with carbon dioxide. The inoculated beetles were placed individually into plastic film roll canisters kept in an environmental chamber at 24 ◦C under a 16 L:8 D photoperiod and fed with fresh apple.

During the following day, we infected the beetles with the conidia of entomopathogenic fungi, *M. robertsii* [19]. To infect the insects with the fungi, we anesthetized beetles with carbon dioxide. We dropped dorsally 5 μL of LD50 solution containing conidium (5 × 106 conidium/mL) with a pipette on the abdomen under the beetles' wings. LD50 doses were determined in preliminary experiments by infecting a separate group of insects with di fferent doses of conidium and selecting the dose that closest to kill 50% of the treated animals. Unfortunately, the mortality caused by the fungi was much higher in the experiment than in our preliminary studies (probably because wounding by the needle made it easier for the fungi to penetrate the cuticula). In our previous studies on *T. molitor*, we found that juvenile hormone administration did not influence on beetles' survival [8]. There was no mortality among beetles when dipped in the control solution. Thus, we left the control solution out from this experiment to double the sample size in the fungal treatment groups. After the infection with fungi, beetles were placed individually back to the plastic film roll canisters in an environmental chamber at 24 ◦C under a 16 L:8 D photoperiod and fed with fresh apple for 21 days to check daily for mortality rates of experimental individuals.
