Prospecção de vírus de RNA para o desenvolvimento de vetor de silenciamento em Euschistus heros (Hemiptera: Pentatomidae)

Abstract

The neotropical brown stink bug, Euschistus heros (F.) (Hemiptera: Pentatomidae), is a major phytophagous insect pest that causes considerable economic losses in soybean crops. Due to the sudden increase in the economic importance of this pest, there are limited management strategies currently available, mostly relying on highly toxic wide-spectrum insecticides. For this reason, there is a strong dependence on the use of non-selective and highly toxic broad-spectrum insecticides that can persist in the environment and affect non-target organisms. Thus, it is crucial to develop new tools, which are efficient and reduce the environmental impact. In this way, the RNA interference (RNAi) technique represents has potential for the development of new insect management strategies. There are several applications of RNAi in insect management, by means of transformative or non-transformative strategies. The virusinduced gene silencing (VIGS) is among the non-transformative strategies. This strategy is based on a genetically modified virus for target gene silencing after insecthost infection. In order to develop solutions based on VIGS, it is necessary to carry out the identification, molecular characterization, biological characterization and construction of an infectious clone of a virus species capable of infecting and replicating the target insect. In this context, the objective of this work is to identify and characterize an RNA virus in E. heros with potential use in the construction of a VIGS vector. For this purpose, the sequencing of E. heros transcriptome and subsequent assembly and identification of viral transcripts was performed. The viral genome with the highest number of transcripts per million was selected for the molecular characterization, for identification of distribution in the productive regions of Brazil and for the construction of an infectious clone enabling more in-depth studies of its biology and subsequent development of a vector of VIGS. Five virus species were identified, amongst them the Halyomorpha halys virus (HhV) (Picornavirales: Iflaviridae) represented 99.9% of the absolute viral transcripts count, in this way, the HhV was selected for the next steps. HhV is a + ssRNA virus with approximately 9 kb in length, composed of 5 'UTR, a polyprotein ORF and a 3'UTR containing a poly A tail. The study of its distribution indicated that there is a pattern of occurrence or difference in viral load between the populations of E. heros from the south and from the central-west and southeast regions of Brazil. It was possible to amplify the HhV virus genome in 4 fragments, of which the last three were effectively cloned into plasmids. The results indicate that HhV is potential candidate for the development of a VIGS vector in E. heros, however, further experiments are needed to elucidate its distribution, host amplitude and to construct an infectious clone, which will be the necessary base to characterize the biology of the virus and to develop a VIGS vector in E. heros.