Sex-Specific Screening Mitigates Basal Metabolic Dimorphism Bias and Reveals DIV1 Survival-Associated Metabolic Signatures in Macrobrachium rosenbergii.

Decapod Iridescent Virus 1 (DIV1) causes severe economic losses in Macrobrachium rosenbergii farming. Serum metabolic biomarkers are valuable tools for disease-resistant breeding, yet most existing studies employ mixed-sex samples, and the impact of sexual dimorphism on screening reliability remains poorly quantified. In this study, non-targeted serum metabolomics was performed on 28 healthy M. rosenbergii (14 females and 14 males), which were divided into marginally resistant and susceptible groups after DIV1 challenge. We compared results from mixed-sex and sex-specific screening strategies, and separately analyzed the overlap of upregulated and downregulated differential metabolites. Significant basal sexual dimorphism was observed in serum metabolism. Mixed-sex analysis identified 247 differential metabolites, while male-specific and female-specific analyses identified 121 and 161, respectively. Only 26 metabolites showed consistent directional changes across all three comparisons, accounting for 10.5% of total mixed-sex differential metabolites. Nearly 90% of mixed-sex results were either sex-specific signals or independent of sex. Analyses that do not account for sex introduce a large number of sex-unrelated differential markers and mask sex-specific metabolic features, and sex-specific screening is a necessary strategy to obtain reliable metabolic biomarkers for crustacean disease-resistant breeding.