The Piranha Genome Provides Molecular Insight Associated to Its Unique Feeding Behavior

GBE The Piranha Genome Provides Molecular Insight Associated to Its Unique Feeding Behavior Manfred Schartl1,2,3,4,*, Susanne Kneitz1, Helene Volkoff5,6, Mateus Adolfi1, Cornelia Schmidt1, Petra Fischer1, Patrick Minx7, Chad Tomlinson7, Axel Meyer8, and Wesley C. Warren7,9 1 Physiologische Chemie, Biozentrum, University of Würzburg, Germany Comprehensive Cancer Center Mainfranken, University Clinic Würzburg, Germany 3 Hagler Institute for Advanced Study, Texas A&M University 4 Department of Biology, Texas A&M University 5 Department of Biology, Memorial University of Newfoundland, St John’s, Canada 6 Department of Biochemistry, Memorial University of Newfoundland, St John’s, Canada 7 McDonnell Genome Institute, Washington University School of Medicine 8 Chair in Zoology and Evolutionary Biology, University of Konstanz, Germany 9 Bond Life Sciences Center, University of Missouri *Corresponding author: E-mail: . Accepted: June 27, 2019 Data deposition: Sequences have been deposited at NCBI under BioProject ID PRJNA533530. Abstract The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas’ feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms. Key words: whole-genome sequencing, genome annotation, comparative genomics, RNA-seq transcriptome, energy homeostasis, starvation. Introduction Piranhas are well-known South-American fishes and paradigmatic representatives of Characids. Their widespread reputation comes from their predatory habits and remarkable feeding adaptations, which include large, sharp teeth, and the strongest bite force determined in any fish to date (Grubich et al. 2012). Red-bellied piranha (Pygocentrus nattereri) (fig. 1) inhabits neotropical freshwater rivers of northeastern Brazil, and the Paraguay and Parana basins. Previous research on this species has primarily focused on attributes related to diet/ feeding habits (Pauly 1994) and social and feeding behavior (Foxx 1972; Sazima and Machado 1990). Although it is mostly a carnivorous piscivore, it can be opportunistic and feed on plants, insects, worms, and crustaceans. Furthermore, it preys upon sick and injured fishes and scavenges on cadavers of fishes and other vertebrates (Pauly 1994). The popular man-eating reputation of piranhas is likely due to the necrophagous habits of these fish (Sazima and de Andrade Guimar~aes 1987), as only rarely have “feeding frenzy” attacks on large live prey have been observed. In the wild, red piranha exhibit social behavior and ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 11(8):2099–2106. doi:10.1093/gbe/evz139 Advance Access publication July 8, 2019 2099 2 GBE Schartl et al. Materials and Methods Genome Assembly and Annotation The red piranha DNA used for sequencing was derived from a single female (Collection ID: Pna-1) among a maintained laboratory population. Using the predicted 1.6 Gb genome size estimate of the white piranha (Carvalho et al. 2002), 2100 FIG. 1.—The sequenced red-bellied Piranha, Pygocentrus nattereri, female. total raw sequence coverage of Illumina reads was 76 (short overlapping reads, 3 kb, 8 kb, and 40 kb matepaired libraries). The overlapping sequence reads (250 bp) were assembled using DiscoVar de novo (Weisenfeld et al. 2014) with default parameters. These reads are derived from a 400 bp library, thus making both pairs overlap in a region of 50 bp, a feature that is exploited by the assembler. This initial assembly of contigs was iteratively scaffolded with long paired reads (3 and 8 kb) using the program SSPACE (Boetzer et al. 2011) and gap filled with a version of Image (Tsai et al. 2010) modified for large genomes. All contigs and scaffolds were cleaned of contaminating sequences by performing a MegaBLAST (Zhang et al. 2000) of the contigs against adapter, bacterial and other vertebrate databases. The final assembly P. nattereri 1.0.2 was masked and then annotated for gene content using the NCBI pipeline described at http://www.ncbi.nlm.nih.gov/books/NBK169439/ . Repeats and transposable elements (TEs) were identified using RepeatModeler (version 1.0.11, http://www.repeatmasker.org/RepeatModeler/, default parameters). To screen the piranha genome for TEs, the resulting TE library was used as input for RepeatMasker (version 4.0, http://repeatmasker.org/). Characteristics of the repeat landscape (including element age and diversity) were plotted using the RepeatMasker perl script “createRepeatLandscape.” Positive Selection To estimate genes under positive selection in piranha the protein and cDNA fasta files for several well-annotated species of fish representing the whole fish tree of life were downloaded from NCBI (supplementary table 1, Supplementary Material online). Orthologous proteins of all fish were identified using inparanoid (O’Brien et al. 2005)with default settings. For each gene with a protein ortholog across all species (comparison 1: n ¼ 11,501, comparison 2: n ¼ 3,949), the corresponding protein and cDNA sequences were aligned and converted Genome Biol. Evol. 11(8):2099–2106 doi:10.1093/gbe/evz139 Advance Access publication July 8, 2019 swim in schools, usually of 20–30 fish that feed together (Bellamy 1968; Sazima and Machado 1990). They engage in acoustic communication, which is sometimes exhibited along with aggressive behaviors (Millot et al. 2011). Red piranha mostly ambush fish from within the aquatic vegetation and dash after passing preys, and scan and pick the sediment while foraging on vegetation or invertebrates (Foxx 1972; Sazima and Machado 1990). They can endure long periods of prey shortage and starvation, in particular, when water levels drop and prey becomes scarce at the end of the summer. Most fish in the wild are faced with s (...truncated)