Bluegill sunfish (Lepomis macrochirus) are one of the classic systems for studying male alternative reproductive tactics (ARTs) in teleost fishes. In this species, there are two distinct life histories: parental and cuckolder, encompassing three reproductive tactics, parental, satellite, and sneaker. The parental life history is fixed, whereas individuals who enter the cuckolder life history transition from sneaker to satellite tactic as they grow. For this study, we used RNAseq to characterize the brain transcriptome of the three male tactics and females during spawning to identify gene ontology (GO) categories and potential candidate genes associated with each tactic. We found that sneaker males had higher levels of gene expression differentiation compared to the other two male tactics. Sneaker males also had higher expression in ionotropic glutamate receptor genes, specifically AMPA receptors, compared to other males, which may be important for increased spatial working memory while attempting to cuckold parental males at their nests. Larger differences in gene expression also occurred among male tactics than between males and females. We found significant expression differences in several candidate genes that were previously identified in other species with ARTs and suggest a previously undescribed role for cAMP-responsive element modulator (crem) in influencing parental male behaviors during spawning.
In June 2013, bluegill sunfish were collected via dip net from Lake Opinion near Queen’s University Biological Station (QUBS), Elgin, Ontario, Canada. A total of 12 parental males, 12 sneaker males, 13 satellite males, and 12 females were collected on the same day directly from the bluegill colony while in the act of spawning. All spawning fish used in this study were behaviorally verified as to tactic by snorkelers prior to collection. An additional 12 non-nesting parental males were collected off of the colony four days prior to spawning (as determined once spawning at these colonies began) and were used as our non-spawning parental males. These males were reproductively mature but were in between spawning bouts. Individuals were euthanized using clove oil, total body length was measured, and whole brains were immediately dissected out and stored in RNAlater (Life Technologies, Carlsbad, CA). The total amount of time required for euthanasia, brain dissection, and brain storage in RNAlater was under 5 minutes. Brains remained in RNAlater at 4°C for 24 hours and were then transferred to fresh cryovials, flash frozen, and kept in liquid nitrogen until they were transported on dry ice to the University of Western Ontario. Samples were then stored at -80°C until total RNA extraction. The Animal Care Committee at Western University (UCC) approved all procedures performed in this study (AUP # 2010–214).
Total RNA Extraction
Total RNA was extracted from whole brains using a standard Trizol (Life Technologies, Carlsbad, CA) extraction protocol (https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf). Total RNA was submitted to the London Genomics Center at the University of Western Ontario and quality was assessed using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Four individuals from each group (spawning parental males, non-spawning parental males, sneaker males, satellite males, and females), for a total of 20 individuals, were submitted to the Michigan State University Research Technology Support Facility—Genomics Center for cDNA library construction and sequencing. Individuals used for this study had RIN (RNA Integrity Number) values ranging from 9.2–9.9.
cDNA Library Construction and Sequencing
The cDNA libraries were constructed for each individual using Illumina TrueSeq Stranded mRNA Library Preparation Kits LT (Illumina, San Diego, CA), with each individual receiving a uniquely identifiable index tag. The quality of each library was evaluated and the 20 individuals were multiplexed into a single sample that was subsequently run on two lanes of an Illumina HiSeq2500 Rapid Run flow cell (v1). Sequencing was performed on paired end 2 x 150 bp format reads and bases were called using Illumina Real Time Analysis software (v22.214.171.124). Reads from each individual were identified based on their unique index tag, separated, and converted to fastq files using Illumina Bcl2fastq v1.8.4. Sequencing produced an average of 14.5 million reads per individual, with over 90% of the reads having a Q-score >30.