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. 2025 Feb 24;26(1):192.
doi: 10.1186/s12864-025-11392-5.

Genetic and metabolic insights into sexual dimorphism in the flexor carpi radialis of Asiatic toads (Bufo gargarizans) associated with amplexus behavior

Hui Ma  1   2 , Long Jin  1   2 , Li Zhao  1   2 , Chengzhi Yan  3   4 , Zhiping Mi  5   6
Affiliations

Genetic and metabolic insights into sexual dimorphism in the flexor carpi radialis of Asiatic toads (Bufo gargarizans) associated with amplexus behavior

Hui Ma et al. BMC Genomics. .

Abstract

Background: Sexual dimorphism, a widespread phenomenon across the animal kingdom, encompasses differences between sexes in size, morphology, and physiological traits. In this study, we investigated sexual dimorphism in the flexor carpi radialis (FCR) muscle, which is critical for amplexus in Asiatic toads (Bufo gargarizans), using integrated transcriptomic and metabolomic approaches.

Results: Male toads exhibited significantly larger FCR muscles, reflecting enhanced muscle function required for sustained amplexus. Transcriptomic analysis identified 818 differentially expressed genes (DEGs) between sexes, with 389 upregulated and 429 downregulated in males, predominantly associated with muscle contraction, sarcomere organization, and energy metabolism. Metabolomic profiling revealed 69 differentially expressed metabolites (DEMs), with male-biased enrichment in pathways involved in protein synthesis and degradation, energy metabolism, and material transport. Integrated analysis pinpointed key metabolic pathways-such as glycine, serine, and threonine metabolism; alanine, aspartate, and glutamate metabolism; fatty acid degradation; and the tricarboxylic acid (TCA) cycle-as central to the observed sexual dimorphism. Among these, the genes AGXT, ACADL, ACAT1, MDH2, and SUCLG2 emerged as pivotal regulators.

Conclusions: Collectively, these findings provide novel insights into the genetic and metabolic basis of sexual dimorphism in B. gargarizans, offering a deeper understanding of the evolutionary mechanisms driving sex-specific traits in vertebrates.

Keywords: Amplexus; Metabolomic; Muscle; Sexual dimorphism; Transcriptomic.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of China West Normal University (approval code: CWNU2021D006). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
(A) and (B) Illustration of the location and sexual dimorphism of the flexor carpi radialis (FCR) in B. gargarizans from a ventral view, with the FCR shown within the white dashed box. (C) and (D) Comparative analysis of FCR wet and dry mass between males and females, independent of SVL (n = 9). Significant differences are indicated by P < 0.001
Fig. 2
Fig. 2
(A) Principal component analysis (PCA) score plot distinguishing male and female B. gargarizans, showing clear clustering between the two sexes. (B) Volcano plot illustrating the genes that are expressed differentially ((DEGs)) in males relative to females. Red and blue dots represent significantly upregulated and downregulated genes, respectively. DEGs were defined by a threshold of|log₂FC| ≥ 2 and an adjusted q-value < 0.05 (n = 9). (C) Gene Ontology (GO) enrichment analysis and (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the identified DEGs. (E) qRT-PCR validation of five DEGs (ALKAL2, MYBPC3, CA6, GPR149, HAL), consistent with RNA-seq results
Fig. 3
Fig. 3
(A) PCA and (B) OPLS-DA score plots differentiating male and female B. gargarizans, showing clear clustering between the two sexes. (C) Volcano plot illustrating the differentially expressed metabolites (DEMs) of males relative to females. Red and blue dots represent significantly upregulated and downregulated metabolites, respectively. (D) Heat map of DEMs in male and female B. gargarizans. Color intensity corresponds to metabolite concentration, with red indicating higher levels and blue indicating lower levels. DEMs were identified based on VIP > 1 and P < 0.05 (n = 9). (E) KEGG pathway enrichment analysis of the identified DEMs
Fig. 4
Fig. 4
(A) Correlation network analysis of DEGs and DEMs. Yellow lines represent positive correlations between DEGs and DEMs, while blue lines represent negative correlations. Blue labels indicate gene nodes, and black labels indicate metabolite nodes. (B) Venn diagram showing the overlap of pathways mapped to both DEGs and DEMs. (C) The glycine, serine, and threonine metabolism pathway, regulated by the DEG AGXT
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