**1. Introduction**

The immunocompetence handicap hypothesis [1] suggests that the expression of secondary sexual traits honestly signals male quality because testosterone—needed to develop these traits—has immunosuppressive e ffects. However, many studies in vertebrates have failed to reveal a clear relationship between the expression of secondary sexual ornamentation and immune defense. This might be because the physiological relationship between these traits is not as simple as originally thought and because of the alterations of stress hormones, which may involve trade-o ffs between sexual ornamentation and immunity [2]. However, many studies in insects and spiders have found that the expression of males' secondary sexual characteristics correlates positively with their immune defense [3–7]. Since insects lack male-specific hormones such as testosterone, it has been suggested that in these animals, the immunocompetence handicap mechanism would be mediated by juvenile hormone (JH) [8].

Juvenile hormone is synthesized in the corpora allata, and it is known to play a crucial role in many aspects of development, reproduction, aging, and behavior in insects. For example, it seems that juvenile hormone type III is associated with sex pheromone production in cockroaches [9]. Similarly, it was found that the administration of juvenile hormone increased the attractiveness of male pheromones in the mealworm beetle, *Tenebrio molitor*. Still, it reduced the strength of the encapsulation response and phenoloxidase activity of hemolymph [8]. Interestingly, it has been found that while JH increases male attractiveness, it reduces the size of the testis and sperm viability, suggesting another cost of high juvenile hormone levels [10]. In the territorial damselfly, *Calopteryx virgo*, it has been found that the administration of methoprene acid (an analog of JH) increased aggression and occupation time in territories but decreased phenoloxidase activity of hemolymph [11]. Juvenile hormone has also been shown to affect genes related to antibacterial peptide expression [12,13]. In the diamondback moth, *Plutella xylostella*, it was shown that juvenile hormone inhibited hemocyte-spreading behavior [13], suggesting that JH induces immune suppression because effective hemocyte-spreading is important for phagocytosis, nodulation, and encapsulation [14]. Furthermore, they found that pyriproxyfen (a JH analog) enhanced the pathogenicity of *Bacillus thuringiensis* subsp. *kurstaki* [13].

Juvenile hormone may also be associated with a trade-off between reproduction and immunity in insects. For example, mating reduced the activity of phenoloxidase enzyme in the hemolymph of both sexes of *T. molitor* [15], but there was no effect on the hemocyte load. It has been found that the observed decrease in phenoloxidase levels was caused by an increase in juvenile hormone levels due to mating, which indicates that juvenile hormone might indeed mediate the trade-off between mating and immunity [15]. Previously, it was shown that mating enhances resistance against entomopathogenic fungi, *Beauveria bassiana,* infection and that the effect was stronger in males than among females of *T. molitor* [16]. This shows that the effect of juvenile hormone on the immune system may be more complicated than previously thought. Overall, these results sugges<sup>t</sup> that studies testing the effect of juvenile hormone on the resistance against highly virulent parasites and pathogens are needed.

Entomopathogenic fungi (EPF), including entomophthoralean fungi, offer environmentally friendly alternatives to conventional synthetic chemicals for arthropod pest control. There are over 750 different species of EPF identified so far [17]. Although entomophthoralean fungi are highly efficacious, much attention has focused on researching species belonging to the order Hypocreales because they are more amenable for mass production and have a relatively wide host range. Approximately 80% of the commercially available EPF products are based on the *Metarhizium robertsii, M. anisopliae, M. brunneum,* and *B. bassiana*.

The study aimed to test whether juvenile hormone affects the resistance of *T. molitor* against a real pathogen and whether there is a sex difference in the pathogen resistance. We tested juvenile hormone-related effects on the immunity of *T. molitor* against *M. robertsii*.

#### **2. Materials and Methods**
