Comparative Genomics and the Salivary Transcriptome of the Redbanded Stink Bug Shed Light on Its High Damage Potential to Soybean

GBE Comparative Genomics and the Salivary Transcriptome of the Redbanded Stink Bug Shed Light on Its High Damage Potential to Soybean Hunter K. Walt1, Jonas G. King1, Tyler B. Towles 1 2 , Seung-Joon Ahn1, Federico G. Hoffmann 1,3, * Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, 2 Macon Ridge Research Station, Louisiana State University, Winnsboro, LA 71295, USA 3 Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Mississippi State, MS 39762, USA *Corresponding author: E-mail: . Accepted: June 05, 2024 Abstract The redbanded stink bug, Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), is a significant soybean pest in the Americas, which inflicts more physical damage on soybean than other native stink bugs. Studies suggest that its heightened impact is attributed to the aggressive digestive properties of its saliva. Despite its agricultural importance, the factors driving its greater ability to degrade plant tissues have remained unexplored in a genomic evolutionary context. In this study, we hy pothesized that lineage-specific gene family expansions have increased the copy number of digestive genes expressed in the salivary glands. To investigate this, we annotated a previously published genome assembly of the redbanded stink bug, per formed a comparative genomic analysis on 11 hemipteran species, and reconstructed patterns of gene duplication, gain, and loss in the redbanded stink bug. We also performed RNA-seq on the redbanded stink bug’s salivary tissues, along with the rest of the body without salivary glands. We identified hundreds of differentially expressed salivary genes, including a subset lost in other stink bug lineages, but retained and expressed in the redbanded stink bug’s salivary glands. These genes were significantly enriched with protein families involved in proteolysis, potentially explaining the redbanded stink bug’s heightened damage to soybeans. Contrary to our hypothesis, we found no support for an enrichment of duplicated digestive genes that are also dif ferentially expressed in the salivary glands of the redbanded stink bug. Nonetheless, these results provide insight into the evo lution of this important crop pest, establishing a link between its genomic history and its agriculturally important physiology. Key words: Piezodorus guildinii, gene family evolution, Hemiptera, gene loss, gene duplication, differential retention. Significance The redbanded stink bug, an important soybean pest in the Americas, inflicts greater damage to soybean due to its high er salivary digestion compared to other stink bugs. We employed comparative genomics and analyses of the salivary transcriptome to explore this and found that the differential retention of ancestral salivary genes may explain this phe nomenon better than gene gains or duplications in the redbanded stink bug genome. We identified a distinct set of genes in the salivary gland that were differentially retained and expressed in the redbanded stink bug lineage, demon strating enrichment in proteolytic function. This discovery offers a potential explanation for the redbanded stink bug’s elevated damage to soybean crops. © The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact . Genome Biol. Evol. 16(7) https://doi.org/10.1093/gbe/evae121 Advance Access publication 12 June 2024 1 MS 39762, USA GBE Walt et al. Introduction Results Genome Annotation We identified 62.09% of the redbanded stink bug genome (GenBank: GCA_023052935.1) as repetitive elements. These regions were masked, and we annotated the masked genome using the BRAKER2 pipeline. We compared all pre dicted proteomes from RNA-seq data and protein data be fore and after the TSEBRA transcript selection (see Materials and Methods) and found the highest BUSCO score with the genome annotation using RNA-seq hints and Augustus gene prediction. We ran the supplementary BRAKER script, selectSupportedSubsets.py, to filter the predicted genes by any external support. This resulted in 13,563 annotated genes with support, of which 12,641 (93.2%) have func tional annotations. We ran the BUSCO analysis in the pro tein mode using the hemiptera_odb10 database and found that 2,144 universal single-copy orthologs out of 2,510 (85.4%) were present in the final protein data set. Comparative Genomics We used the 11 hemipteran genomes that met our thresholds for our comparative genomics analysis (supplementary table S1, Supplementary Material online). A total of 196,495 genes were assigned to 17,545 orthogroups by the OrthoFinder al gorithm. We found 1,339 single-copy orthologs shared be tween the 11 species and built a species tree based on a concatenated alignment of the single-copy orthologs (Fig. 2). 2 Genome Biol. Evol. 16(7) https://doi.org/10.1093/gbe/evae121 Advance Access publication 12 June 2024 The redbanded stink bug, Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), is an important soybean (Glycine max (L.) Merr.) pest in the Americas with a distinct ability to digest plant tissues. It was first described in 1837 on the island of St. Vincent in the Caribbean, and although its geographic origin remains unclear, studies have suggested that it originated in the Caribbean Basin and reached Brazil about a million years ago (Moraes et al. 2023). More recently, the redbanded stinkbug expanded its range and population size in conjunction with increases in soybean production across South America (Bundy et al. 2018; Zucchi et al. 2019; Moraes et al. 2023). Now, it ranges from Argentina through Central America, Mexico, and the southern United States from Texas to South Carolina (Sosa-Gómez et al. 2020). It has been consistently designated as an economically significant pest in Brazil, Argentina, and Uruguay since the 1970s (more recently in the United States), and it is highly capable of expanding its range (Bundy et al. 2018; Chen et al. 2023). Interestingly, other species of the genus Piezodorus are pests of legume crops in Europe, Asia, Africa, and Australia, but the redbanded stink bug is the only species that exists in the New World, with its closest relatives found in Australia (Piezodorus oceanicus), Asia, and Africa (Piezodorus hybneri) (Bundy et al. 2018) (supplementary fig. S1, Supplementary (...truncated)