Abstract:Sex determination and gonadal development in fish are highly plastic processes coordinated by genetic and environmental factors, providing a foundation for sex-directed breeding in aquaculture. Largemouth bass (Micropterus salmoides), a native North American species, is a globally valuable freshwater aquaculture fish due to its rapid growth, delicious flesh, and lack of intermuscular bones. However, female largemouth bass exhibit a significantly higher gonadosomatic index (GSI) (peak up to 16.0%) than males, directly reducing the net meat rate and commercial quality. Thus, establishing all-male breeding systems is critical for optimizing industry economic benefits. To date, research on largemouth bass sex determination has focused on transcriptional regulation, confirming its XX/XY system and identifying key genes like dmrt1 and cyp19a1b. Yet, post-transcriptional regulation by microRNAs (miRNAs) remains understudied. miRNAs are 20–24 nt endogenous non-coding RNAs that mediate post-transcriptional regulation by binding target mRNA 3" untranslated regions, inhibiting translation or inducing degradation. Studies in Nile tilapia, Atlantic halibut, and golden pompano have shown sex-differentially expressed miRNAs regulate sex determination by targeting core sex-related genes. This study aimed to investigate gonadal miRNA expression profiles in largemouth bass, explore their regulatory mechanisms, and screen sex identification markers. 24 sexually mature (1-year-old) largemouth bass (12 females, 12 males) were obtained from Guangdong Liangshi Aquatic Seed Industry Co., Ltd. After MS-222 anesthesia, gonadal tissues were collected, with each sex grouped into 3 biological replicates (4 fish/replicate) for small RNA library construction. Total RNA was extracted via TRIzol, and 18–30 nt small RNA fragments were isolated by polyacrylamide gel electrophoresis. Following adapter ligation, reverse transcription, and PCR amplification, ~140 bp libraries were sequenced on the Illumina X-ten platform. Raw reads were filtered to remove adapters, short inserts (<18 nt), and polyA-enriched reads (A content >80% or consecutive A ≥10). Clean data were aligned to GenBank/Rfam databases to exclude non-target RNAs, then mapped to the largemouth bass reference genome (GenBank ID: GCA-019677235.1) for miRNA identification. Differential expression analysis was performed with edgeR (p < 0.05, |log2FC| > 2). Target genes were predicted using Miranda, TargetScan, and RNAhybrid (intersection + binding free energy ≤ -20 kcal/mol), followed by GO/KEGG enrichment analyses (p < 0.05). Eleven random differential miRNAs were validated via qRT-PCR (U6 as internal reference). Four core miRNA-target gene pairs (targeting sex-determining genes) were verified in control (XY-M, XX-F) and sex-reversed (XY-F, XX-M) groups by qRT-PCR (β-actin as reference, 2-△△Ct method). High-throughput sequencing yielded 80.3 million raw reads, with 11.9 million clean tags per library (Q20 ≥ 98.2%). The average miRNA length was 22 nt, consistent with typical animal miRNAs. A total of 1109 sexually dimorphic miRNAs were identified (610 known, 499 novel), including 477 significantly differential miRNAs (273 female-upregulated, 204 male-upregulated), 53 female-specific, and 78 male-specific miRNAs. Target genes included sex-related regulators (dmrt1, cyp19a1b, amh, piwil1). GO enrichment showed target genes involved in cellular/metabolic processes, catalytic activity, and binding; KEGG enrichment indicated enrichment in ErbB signaling, cytokine-cytokine receptor interaction, programmed necrosis, and endoplasmic reticulum protein processing. qRT-PCR confirmed sequencing reliability. The 4 core pairs (miR-17-x-dmrt1, miR-221-y-piwil1, miR-725-z-sox9a, miR-190-x-amh) showed female-high miRNA expression and male-high target gene expression (p < 0.05), with significant differences between sex-reversed groups, distinguishing genetic and phenotypic sexes. This study first systematically reveals largemouth bass gonadal miRNA expression profiles, filling gaps in post-transcriptional sex determination research. Female/male-specific miRNAs enrich sex-related molecular targets, and pathway enrichment reflects the complex miRNA regulatory network. The 4 core pairs are promising sex identification markers, enabling rapid screening for all-male population construction to address high female GSI issues. These findings deepen understanding of largemouth bass sex determination and provide molecular targets for sex-directed breeding. Future research will validate core miRNA functions via gene silencing/overexpression to clarify their roles in sex determination.
