Involvement of laccase2 and yellow-e genes in antifungal host defense of the model beetle, Tribolium castaneum

We previously reported that the moderate knockdown of chitin synthase 1 gene of the model beetle Tribolium castaneum impairs the host defense against entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, which infect host insects via the direct penetration of cuticular integuments (Hayakawa et al., 2017). In this study, we focused on the antifungal roles of laccase2 (Lac2) as well as yellow-e (Y-e) genes, both of which are shown to be important to the establishment of stable cuticular structures in this beetle species. The expression profiles of the two genes somewhat resembled each other, peaking in late prepupae and mid to late pupae, while the transcript levels of Lac2 were higher than Y-e throughout. The knockdown of Lac2 gene at the prepupal and pupal peaks with relatively small amounts of dsRNA resulted in pupae with a lighter color and adults with a lighter color and dimpled/wrinkled elytra, respectively. Meanwhile, similar gene knockdown of Y-e but with 10 times more dsRNA compared to Lac2 resulted in pupae having a normal appearance and adults with a darker color. We conducted fungal infection assays with B. bassiana and M. anisopliae using these knockdown animals. The knockdown of Y-e gene had no or limited effects in both pupae and adults in terms of the antifungal host defense. Similarly, the knockdown of Lac2 gene did not change significantly the defense phenotypes of the resulting pupae. By sharp contrast, the host defense of the adult beetles against the two fungal species was almost totally destroyed by the moderate knockdown of Lac2 gene, suggesting its indispensable role in antifungal host defense presumably through the construction of sound cuticles of the adults. Finally, we investigated the maturation of host defense against fungal infection in the Lac2 knockdown adults and found that while the day 10 adults were still susceptible to M. anisopliae infection with some delay of death in comparison with day 1 adults, they exhibited complete refractoriness to B. bassiana.