Ecotype differences in aggression, neural activity and behaviorally relevant gene expression in cichlid fish

Purpose: The goal of this study is to identify genes that showed a pattern of differential expression between species within activated neurons during mirror-elicited aggression between two species of Lake Malawi cichlid fish which differ in aggressive behaviors, Mchenga conophoros (MC, sand-dwelling) and Petrotilapia chitimba (PC, rock-dwelling). To do this, we use antibodies to phosphorylated ribosomal protein S6 (pS6) to enrich for transcripts bound to phosphorylated ribosomes followed by RNA-seq (PhosphoTRAP). In neurons, this phosphorylation occurs downstream of the binding of neurotransmitters and, thus, pS6 antibodies are increasingly being used as markers of neural activity, similar to immediate early genes (IEGs) like c-fos or egr-1. Methods: Whole brain mRNA samples were collected from 8 adult male cichlids (4 MC, 4 PC) 90min following either 15min exposure to mirror in home tank (Mirror Test) or a Control condition. RNA-immunoprecipitation using antibodies to pS6 were performed and RNA was extracted from immunoprecipitated (IP) RNA samples. We also collected total RNA (input), prior to immunoprecipitation. For details see extraction protocol below. RNA from both input and IP samples was reverse transcribed using the SMART-Seq v4 Ultra Low Input RNA kit. Libraries were prepped using the NEBNext Ultra II FS DNA library Prep Kit for Illumina and sequenced. Sequence reads that passed quality filters were aligned to the Metriaclima zebra (Malawi cichlid) reference genome UMD2A using HiSat2. Transcripts were quantified using StringTie and differential expressional analyses were performed using DESeq2. Results: We identified a large number of genes showing differential expression in mirror-elicited aggression, as well as many genes that differ between ecotypes. These genes, which may underly species differences in behavior, include several neuropeptides, genes involved in the synthesis of steroid hormones, and neurotransmitter activity. Conclusions: This work lays the foundation for future experiments using this emerging genetic model system to investigate the genomic basis of evolved species differences in both brain and behavior. Overall design: Whole brain mRNA samples were collected from 8 adult male cichlids (4 of each species: Mchenga conophoros, MC, and Petrotilapia chitimba, PC), 90min following either 15min exposure to mirror in home tank (Mirror Test) or a Control condition. MC and PC differ in mirror-elicited aggression. Samples were processed in two batches, each containing all four conditions, all collected and processed together on the same day. Immunoprecipitation using antibodies to phosphorylated ribosomal protein S6 (pS6) was used to enrich for transcripts from activated neurons (PhosphoTRAP). We compared input (total RNA) to immunoprecipitated (IP) samples across condition and species to identify behaviorally relevant gene expression and genes exhibit patterns of differential expression across species.