Applied Entomology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
Applied Entomology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
J Invertebr Pathol. 2018 Jan;151:41-49. doi: 10.1016/j.jip.2017.10.010. Epub 2017 Nov 2.
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.
我们之前曾报道过，模式甲虫赤拟谷盗中几丁质合成酶 1 基因的适度敲低会损害宿主对昆虫病原真菌白僵菌和绿僵菌的防御能力，这两种真菌通过穿透宿主昆虫的表皮来感染宿主（Hayakawa 等人，2017）。在这项研究中，我们专注于漆酶 2（Lac2）和黄-E（Y-e）基因的抗真菌作用，这两个基因在该甲虫物种中建立稳定的表皮结构中被证明是重要的。这两个基因的表达谱有些相似，在预蛹后期和中晚期蛹期达到峰值，而 Lac2 的转录水平在整个过程中都高于 Y-e。用相对少量的 dsRNA 在预蛹和蛹高峰期敲低 Lac2 基因，导致蛹的颜色变浅，成虫的颜色变浅，鞘翅出现凹痕/皱纹。同时，与 Lac2 相比，用 10 倍量的 dsRNA 对 Y-e 进行类似的基因敲低，导致蛹的外观正常，成虫的颜色更深。我们使用这些敲低动物进行了白僵菌和绿僵菌的真菌感染实验。Y-e 基因的敲低在蛹和成虫的抗真菌宿主防御方面没有或只有有限的影响。同样，Lac2 基因的敲低也没有显著改变由此产生的蛹的防御表型。相比之下，Lac2 基因的适度敲低几乎完全破坏了成虫对两种真菌的宿主防御能力，这表明它在抗真菌宿主防御中不可或缺的作用，可能是通过构建成虫健全的表皮来实现的。最后，我们研究了 Lac2 敲低成虫对真菌感染的宿主防御成熟过程，发现虽然第 10 天的成虫仍易受绿僵菌感染，但与第 1 天的成虫相比，死亡时间有所延迟，但它们对白僵菌完全具有抵抗力。
