Validation of Thymidylate Synthase as a Key Gene Inhibiting WSSV Proliferation in Procambarus clarkii

Abstract

White spot syndrome virus (WSSV) is a highly pathogenic agent that poses a significant constraint on the sustainable aquaculture of the red swamp crayfish (P. clarkii). Thymidylate synthase (TS) and ribonucleotide reductase (RR), two genes involved in viral DNA replication, are potential targets for RNAi-based control, but their functional validation and low-cost use remain limited. Bioinformatics analysis revealed that WSSV TS differs evolutionarily from crustacean TS but shares 64% homology with P. clarkii TS, suggesting potential virus-host substrate competition. In vitro-synthesized dsRNA-TS and dsRNA-RR both significantly suppressed WSSV replication in infected P. clarkii. TS was selected for further study due to its evolutionary profile and potential compatibility with molecular breeding approaches. The dsRNA-TS injection eliminated detectable virus within 3 days and reduced cumulative mortality by 10%. Under simulated transport stress conditions, dsRNA-TS did not enhance survival rates, likely due to immunosuppressive effects; however, it sustained the suppression of WSSV replication from 7 to 14 days post-infection. The dsRNA-TS expressed in Escherichia coli HT115 (DE3) had no significant effect, probably because of low purity, low concentration, and poor delivery. The findings provide a sustainable biological control strategy against WSSV in P. clarkii aquaculture, and lay the foundation for the optimization of prokaryotic dsRNA production systems as well as the integration of RNAi with molecular breeding techniques.

Keywords

dsRNA / White spot syndrome virus / Procambarus clarkii / Thymidylate synthase / Ribonucleotide reductase

Statement of the Use of Generative AI and AI-Assisted Technologies in the Writing Process

AI solely assisted in completing refining academic language and designing the figure and table formats for the initial draft. The authors independently conducted all core research work and bear full responsibility for the scientific validity and originality of the study.

Author Contributions

Z.Y.: Methodology, Investigation, Writing–original draft, Formal analysis. T.Z.: Methodology, Investigation. K.Y.: Methodology, Investigation. Y.C.: Methodology, Investigation. Y.X.: Methodology, Investigation. Y.Y.: Resources. Y.L.: Project administration, Supervision, Funding acquisition, Writing– review & editing, Resources, Conceptualization.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper.

Funding

This work was supported by the Fundamental Research Funds for the Central Universities (2662025FW008), and Fishery Seed Industry Joint Breeding Program of Jiangxi Province (sczy202501).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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