De Novo Assembly and Characterization of Different Insect Transcriptomes Against Metarhizium Anisopliae (Metschn.) Through Next-generation Sequencing

Abstract

Agricultural pests Hellula undalish and Helicoverpa armigera are significant threats to a wide array of crops, causing substantial damage to agricultural yields. The use of entomopathogenic fungi has emerged as a promising biocontrol approach against these pests, with Metarhizium anisopliae showing remarkable potential as a natural insect pathogen. In this study, the aim was to investigate the transcriptomic responses of H. undalis and H. armigera to Metarhizium anisopliae treatment using high- throughput sequencing and advanced assembly techniques. By analyzing the transcriptome, it aimed to identify key immune-related genes and pathways associated with the host response to the fungal pathogen. To achieve this, H. undalis and H. armigera were collected and exposed to Metarhizium anisopliae and compared them with non-treated control. Total RNA was extracted from the treated and non-treated insect samples and cDNA libraries were constructed. Subsequently, the libraries were sequenced on the Illumina NovaSeq6000 platform, generating a substantial number of raw reads for both treated and non-treated conditions. Before analysis, the raw reads underwent quality control and filtering to ensure data accuracy and reliability. The processed reads were then subjected to de novo assembly, where overlapping reads were assembled into contigs. This assembly process resulted in a comprehensive representation of the transcriptomes for both H. undalis and H. armigera under different conditions. To functionally annotate the transcriptomes, the assembled contigs were compared to known sequences in the NCBI-NR database, allowing us to identify homologous genes and assign putative functions. Additionally, Gene Ontology (GO), Clusters of Orthologous Groups (COG) database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database were utilized to categorize the identified genes based on their molecular functions, biological processes, and involvement in metabolic pathways. The functional annotation revealed significant similarities between the identified sequences and those from various insect species, providing valuable insights into the functional genes shared among insects. Notably, our analysis identified a set of immune-related genes in H. undalis and H. armigera, including those associated with the Toll and IMD signaling pathways. These pathways play crucial roles in the insect's immune response against fungal pathogens. Moreover, significant upregulation of genes involved in the Toll and IMD pathways were observed in response to Metarhizium anisopliae treatment, suggesting a heightened immune response against the fungal pathogen. These finding provides essential information on the host-pathogen interaction dynamics and immune modulation in both insects under entomopathogenic fungi treatment. In conclusion, this study provides first transcriptomic profiles of H. undalis worldwide and H. armigera in India. Furthermore, it also presents the first transcriptomic profile under Metarhizium anisopliae treatment. The identification and functional annotation of immune- related genes, particularly those involved in the Toll and IMD signaling pathways, provide valuable insights into the defense mechanisms of these agricultural pests against entomopathogenic fungi. This knowledge opens new avenues for future research on pest management strategies and immune modulation, contributing to sustainable and effective agricultural practices