Tenebrio molitor Gram‐negative‐binding protein 3 (TmGNBP3) is essential for inducing downstream antifungal Tenecin 1 gene expression against infection with Beauveria bassiana JEF‐007

Abstract

The Toll signaling pathway is responsible for defense against both Gram-positive bacteria and fungi. Gram-negative binding protein 3 (GNBP3) has a strong affinity for the fungal cell wall component, β-1,3-glucan, which can activate the prophenoloxidase (proPO) cascade and induce the Toll signaling pathway. Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in the Toll signaling pathway. In this study, we monitored the response of 5 key genes (TmGNBP3, TmMyD88, and Tenecin 1, 2, and 3) in the Toll pathway of the mealworm Tenebrio molitor immune system against the fungus Beauveria bassiana JEF-007 using RT-PCR. TmGNBP3, Tenecin 1, and Tenecin 2 were significantly upregulated after fungal infection. To better understand the roles of the Toll signaling pathway in the mealworm immune system, TmGNBP3 and TmMyD88 were knocked down by RNAi silencing. Target gene expression levels decreased at 2 d postknockdown and were dramatically reduced at 6 d post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 d post-dsRNA injection. Silencing of TmMyD88 and TmGNBP3 resulted in reduced resistance of the host to fungal infection. Particularly, reducing TmGNBP3 levels obviously downregulated Tenecin 1 and Tenecin 2 expression levels, whereas silencing TmMyD88 expression resulted in decreased Tenecin 2 expression. These results indicate that TmGNBP3 is essential to induce downstream antifungal peptide Tenecin 1 expression against B. bassiana JEF-007.

Evaluation of a cuticle protein gene as a potential RNAi target in aphids

“…insecticides and pathogens) would be desirable in controlling pests as well as for delaying insecticide resistance development . Silencing of immunity genes, Gram‐negative binding protein 3 and Toll receptor reduced the resistance of the insects to fungal infection . In addition to immunity genes, RNAi mediated knock‐down of a cuticle‐related gene encoding chitin synthase A enhanced the virulence of Beauveria bassiana against Spodoptera exigua .…”

“…38,39 Silencing of immunity genes, Gram-negative binding protein 3 and Toll receptor reduced the resistance of the insects to fungal infection. 40,41 In addition to immunity genes, RNAi mediated knock-down of a cuticle-related gene encoding chitin synthase A enhanced the virulence of Beauveria bassiana against Spodoptera exigua. 42 As a key factor in cuticle waterproofing, decreased expression of CPs combined with insect pathogen treatment could be important in the integrated population management of aphids.…”

A constitutively expressed antifungal peptide protectsTenebrio molitorduring a natural infection by the entomopathogenic fungusBeauveria bassiana

“…In the second case, it is likely that the selection pressure exerted by fungal pathogens is constant in the environment of these species of termites, making the evolution of an external constitutive antifungal defense relevant (Bulmer & Crozier, 2004 ;Adler & Karban, 1994 (Lee et al, 1996) might even indicate that this pressure is stagedependent. Since other AMPs are induced in T. molitor following B. bassiana infection through the Toll pathway (Yang et al, 2017), a future lead to follow could be to perform knockdown on these peptides simultaneously, which would shed light on the interplay of constitutive and inducible immune defenses. Our study also adds support to previous observations made in D. melanogaster that the evolution of the pathogen load inside the host over time can show a bimodal outcome, where one category of hosts carry a low level infection while the other category will die of high infection levels (Clemmons et al, 2015 ;see Duneau et al, 2017 for data & review).…”

“…This makes their contribution to insect fitness in case of a natural infection hard to predict, especially regarding peptides whose expression is not elicited by a fungal infection. In the mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae), it has recently been shown that an infection with B. bassiana activated the Toll pathway which lead to the expression of some antimicrobial peptides among such as the defensin Tenecin 1 and the coleoptericin Tenecin 2 (Yang et al, 2017), and that the knock down of this pathway decreased the survival of fungal infected beetles. However, the contribution of the constitutively expressed thaumatin Tenecin 3 (Makarova et al, 2016 ;Johnston et al, 2014 ;Yang et al, 2017) during B. bassiana infection in T. molitor is unknown.…”

“…). In T. molitor, by performing knockdowns of the Toll pathway,Yang et al (2017) highlighted the importance of inducible immune defenses in the survival to B. bassiana, although the authors did not directly knockdown the inducible AMPs suspected to be involved, whereas the role of the constitutive Tenecin 3 was not investigated. Even though the pressure exerted by B. bassiana on T. molitor in its natural habitat is unknown, B. bassiana is widespread in most ecosystems investigated (e.g.…”